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HPMA

HPMA
 
SYNONYMS: HPMA; Hydrolyzed Polymaleic Anhydride; hydrolyzed polymaleic acid; hidrolized polymaleik asit; hidroliz polimaleik asit; HYDROLYZED POLYMALEIC ACID; HIDROLIZED POLYMALEIK ACIT; HİDROLİZED POLİMALEİK ASİT; poly(maleicacid); Polymaleic Acid; MALEIC ACID POLYMER;Polymaleic Acid; HPMA copolymers; Jindřich Kopeček* and Pavla Kopečková; N-(2-hydroxypropyl)methacrylamide), HPMA, N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide; N-(2-Hydroxypropyl) methacrylamide; N-(2 hydroxypropyl); Octanoic acid; Caprylic acid; N-octanoic acid; Octylic acid; N-caprylic acid; N-octylic acid; caprylic acid; caprylic acid; 14C-labeled; caprylic acid; aluminum salt; caprylic acid; ammonia salt; caprylic acid; barium salt; caprylic acid; cadmium salt; caprylic acid; calcium salt; caprylic acid; cesium salt; caprylic acid; caprylic acid, iridum(+3) salt; caprylic acid, iron(+3) salt; caprylic acid, lanthanum(+3) salt;caprylic acid, lead(+2) salt; caprylic acid, lithium salt; caprylic acid, manganese salt; caprylic acid, nickel(+2) salt; caprylic acid, potassium salt; caprylic acid, ruthenium(+3) salt; caprylic acid, sodium salt; caprylic acid, sodium salt, 11C-labeled; caprylic acid, tin salt; caprylic acid, tin(+2) salt; caprylic acid, zinc salt; caprylic acid, zirconium salt; caprylic acid, zirconium(+4) salt; octanoate; octanoic acid; sodium caprylate sodium octanoate; kaprilikasit; caprylicacid; kapirilik asit; caprylicasit; kaprılık asıt; caprylıc acıd;caprylic acid; caprylic acid, 14C-labeled; caprylic acid, aluminum salt; caprylic acid, ammonia salt; caprylic acid, barium salt; caprylic acid, cadmium salt; caprylic acid, calcium salt; caprylic acid, cesium salt; caprylic acid, chromium(+2) salt; caprylic acid, cobalt salt; caprylic acid, copper salt; caprylic acid, copper(+2) salt; caprylic acid, iridum(+3) salt; caprylic acid, iron(+3) salt; caprylic acid, lanthanum(+3) salt; caprylic acid, lead(+2) salt; caprylic acid, lithium salt; caprylic acid, manganese salt; caprylic acid, nickel(+2) salt; caprylic acid, potassium salt; caprylic acid, ruthenium(+3) salt; caprylic acid, sodium salt; caprylic acid, sodium salt, 11C-labeled; caprylic acid, tin salt; caprylic acid, tin(+2) salt; caprylic acid, zinc salt; caprylic acid, zirconium salt; caprylic acid, zirconium(+4) salt; lithium octanoate; octanoate; octanoic acid; sodium caprylate; sodium octanoate; octanoic acid; caprylic acid; 124-07-2; n-octanoic acid; n-caprylic acid; Octylic acid; octoic acid; n-octylic acid; n-Octoic acid; neo-fat 8; 1-heptanecarboxylic acid; Enantic acid; Octic acid; C-8 acid; Caprylsaeure; capryloate; octanoicacid; Kaprylsaeure; Hexacid 898; Acido octanoico; 0ctanoic acid; Acide octanoique; Acidum octanocium; Caprylic acid (natural); Fatty acids, C6-10; Kyselina kaprylova; octylate; Octansaeure; 1-octanoic acid; Acide octanoique [French]; Acido octanoico [Spanish]; Acidum octanocium [Latin]; Kyselina kaprylova [Czech]; NSC 5024; Octanoic acid [USAN:INN]; UNII-OBL58JN025; FEMA No. 2799; CCRIS 4689; HSDB 821; C8:0; Emery 657; Prifac 2901; Lunac 8-95; EINECS 204-677-5; OCTANOIC ACID (CAPRYLIC ACID); BRN 1747180; CH3-[CH2]6-COOH; AI3-04162; caprylic acid, zinc salt; OBL58JN025; CHEBI:28837; caprylic acid, barium salt; caprylic acid, sodium salt; NSC5024; WWZKQHOCKIZLMA-UHFFFAOYSA-N; caprylic acid, cadmium salt; caprilate; Caprylic acid, potassium salt; n-caprylate; caprylic acid, tin(+2) salt; MFCD00004429; n-octoate; n-octylate; FA(8:0); caprylic acid, copper(+2) salt; Octanoic acid, 99%; NCGC00090957-01; C8H16O2; 1-heptanecarboxylate; DSSTox_CID_1645; DSSTox_RID_76259; DSSTox_GSID_21645; OCA; 124-07-2 (Parent); CAS-124-07-2; CH3-[CH2]6-COO(-); caprylic acid, tin salt; caprylic acid, cesium salt; caprylic acid, cobalt salt; caprylic acid, copper salt; caprylic acid, ammonia salt; caprylic acid, calcium salt; caprylic acid, 14C-labeled; caprylic acid, aluminum salt; caprylic acid, manganese salt; caprylic acid, zirconium salt; octanic acid; Caprilic acid; caprylic acid, iron(+3) salt; caprylic acid, lead(+2) salt; acidum octanoicum; octanoate radical; caprylic acid, iridum(+3) salt; caprylic acid, nickel(+2) salt; caprylic acid, chromium(+2) salt; caprylic acid, lanthanum(+3) salt; caprylic acid, ruthenium(+3) salt; caprylic acid, zirconium(+4) salt; EINECS 273-085-7; Acid C8; Octanoic acid radical; Caprylic acid (NF); Kortacid 0899; Neo-Fat 8S; Caprylic Acid 657; Octanoate, ion(1-); caprylic acid, sodium salt, 11C-labeled; n-heptanecarboxylic acid;Octanoic acid (USAN); Fatty acids, C6-1O; ACMC-1BTHQ; Lunac 8-98; AC1Q2VVU; 18312-04-4; C8:0 (Lipid numbers); Heptane-1-carboxylic acid; Octanoic acid, >=98%; Octanoic acid, >=99%; bmse000502; Caprylic/Capric Acid Blend; D0XS4G; EC 204-677-5; SCHEMBL3933; WLN: QV7; NCIOpen2_002902; NCIOpen2_009358; Octanoic acid (USAN/INN); 4-02-00-00982 (Beilstein Handbook Reference); 68937-74-6; KSC174S6D; MLS002415762; Octanoic acid, >=96.0%; Octanoic acid, zirconium salt; Octanoic acid (mixed isomers); AC1L193V; CHEMBL324846; GTPL4585; Octanoic acid, >=98%, FG; QSPL 011; QSPL 184; 557-09-5 (zinc salt); DTXSID3021645; 764-71-6 (potassium salt); CTK0H4961; KS-00000WZB; 4696-54-2 (barium salt); 6700-85-2 (cobalt salt); 2191-10-8 (cadmium salt); 5972-76-9 (ammonium salt); 6028-57-5 (aluminum salt); 6107-56-8 (calcium salt); MolPort-001-792-057; HMS2270A23; Octanoic acid, analytical standard; 16577-52-9 (lithium salt); NSC-5024; ZINC1530416; 1984-06-1 (hydrochloride salt); EINECS 242-197-8; Tox21_111045; Tox21_201279; Tox21_300345; ANW-18188;LMFA01010008; LS-691; SBB060020; STL282742; 7435-02-1 (unspecified Ce salt); AKOS000118802; Octanoic acid, natural, >=98%, FG; Octanoic acid, zirconium salt (1:?); 6535-20-2 (unspecified iron salt); AN-1411; DB04519; MCULE-5193957469; NE10316; Octanoic acid, for synthesis, 99.5%; RTR-003747; 3130-28-7 (iron(+3) salt); 3890-89-9 (copper(+2) salt); 4995-91-9 (nickel(+2) salt); 6427-90-3 (chromium(+2) salt); 7319-86-0 (lead(+2) salt); 15696-43-2 (unspecified lead salt); 1912-83-0 (tin(+2) salt); 5206-47-3 (zirconium(+4) salt);NCGC00090957-02; NCGC00090957-03; NCGC00090957-04; NCGC00254446-01; NCGC00258831-01; 20543-04-8 (unspecified copper salt); HY-41417; M789; SMR001252279; 6535-19-9 (unspecified manganese salt); 20195-23-7 (unspecified chromium salt); 60903-69-7 (La(+3) salt); 67816-08-4 (Ir(+3) salt); 68957-64-2 (Ru(+3) salt); TR-003747; 18312-04-4 (unspecified zirconium salt); 597-EP2269988A2; 597-EP2270008A1; 597-EP2275413A1; 597-EP2287156A1; 597-EP2292617A1; 597-EP2298780A1; 597-EP2301940A1; 597-EP2305689A1; 597-EP2311807A1; 597-EP2311824A1; 597-EP2374787A1; CS-0016549; FT-0660765; O0027; ST51046283; C06423; D05220; 146488-EP2280007A1; 146488-EP2292597A1; SR-01000865607; I04-1204; J-005040; SR-01000865607-2; BRD-K35170555-001-07-9; Z955123584; Octanoic acid, certified reference material, TraceCERT(R); UNII-13FB83DEYU component WWZKQHOCKIZLMA-UHFFFAOYSA-N; UNII-DI775RT244 component WWZKQHOCKIZLMA-UHFFFAOYSA-N; 43FDA9D7-2300-41E7-A373-A34F25B81553; Caprylic acid, European Pharmacopoeia (EP) Reference Standard; UNII-79P21R4317 component WWZKQHOCKIZLMA-UHFFFAOYSA-N; Caprylic acid, United States Pharmacopeia (USP) Reference Standard; 1819997-17-5; 2171005-29-9; Caprylic Acid (Octanoic Acid), Pharmaceutical Secondary Standard; Certified Reference Material ; 124-07-2 [RN]; 1747180 [Beilstein]; 1-Heptanecarboxylic acid; 1-octanoic acid; 204-677-5 [EINECS] 2799; Acid C8; Acide octanoique [French]; Acide octanoïque [French] [INN] [ACD/IUPAC Name]; Acido octanoico [Spanish]; Acidum octanocium [Latin]; caprylic acid [Wiki]; Enantic acid; Liquefied petroleum gas; n-caprylic acid; n-Octanoic Acid; n-Octylic acid; n-undecylic acid; Octanoic acid [ACD/Index Name] [INN] [ACD/IUPAC Name]; Octansäure [German] [ACD/IUPAC Name]; RH0175000; ?????????? ??????? [Russian] [INN]; ??? ?????????? [Arabic] [INN]; ?? [Chinese] [INN]; 4-02-00-00982 [Beilstein]; ácido octanoico [Spanish] [INN]; acidum octanoicum; Capric Acid; Caprinic acid; Capryllic acid; Caprylsaeure; Caprynic acid; Carpylic acid; Heptane-1-carboxylic acid; Hexacid 1095; Hexacid 898; Kaprinsaeure; Kaprylsaeure; Kortacid 0899; Kyselina kaprylova [Czech]; LUNAC 8-95; Lunac 8-98; n-Capric acid; Neo-fat 10 [Trade name]; neo-fat 8; Neo-Fat 8S; n-octic acid; n-Octoic acid; Nuocure 28 [Trade name]; octanoic acid, 98+%; octanoic acid, 99%; octanoic acid, from palm; octanoic acid, reagent; Octic acid; octoic acid; Octylic acid; Prifac 2901; QV7 [WLN]; QV9 [WLN]; capryl; caprylaldehyde; caprylic; caprylic acid; caprylin; caprylyl; caps; 1-heptanecarboxylic acid; Acide octanoïque; Acide octanoique; ácido octanoico; Acidum octanocium; Acidum octanoicum; C8;0; Caprylic acid; Kaprylsäure; n-caprylic acid; n-octanoic acid; n-Octoic acid; n-octylic acid; kaprilik asit, zirkonyum (+4) tuzu; EINECS 273-085-7; Asit C8; Oktanoik asit kökü; Kaprilik asit (NF); Kortacid 0899; Neo-Fat 8S; Kaprilik Asit 657; Oktanoat, iyon (1-); kaprilik asit, sodyum tuzu, 11C-işaretli; n-heptankarboksilik asit; Oktanoik asit (USAN); Yağ asitleri, C6-1O; ACMC-1BTHQ; Lunac 8-98; AC1Q2VVU; 18312-04-4; C8: 0 (Lipid sayıları); Heptan-1-karboksilik asit; Oktanoik asit,> = 98%; Oktanoik asit,> =% 99; bmse000502; Kaprilik / Kaprik Asit Karışımı; D0XS4G; EC 204-677-5; SCHEMBL3933; WLN: QV7; NCIOpen2_002902; NCIOpen2_009358; Oktanoik asit (USAN / INN); 4-02-00-00982 (Beilstein El Kitabı Referansı); 68937-74-6; KSC174S6D; MLS002415762; caprylic acid; caprlic acid; carplic acid; CAPRIYLIC ACID; CARPLIC ACID; CAPLIC ACID; KAPRİLİK ASİT; KAPLİK ASİT; KAPİLİK ASİT; KAPLİK ACİD; CAPRY ACID; CAPY ACID; CAPRYLIC; ACID; acid; asit; caprylic; Octanoic acid; Oktanoik asit,> =% 96.0; Oktanoik asit, zirkonyum tuzu; Oktanoik asit (karışık izomerler); AC1L193V; CHEMBL324846; GTPL4585; Oktanoik asit,> =% 98, FG; QSPL 011; QSPL 184; 557-09-5 (çinko tuzu); DTXSID3021645; 764-71-6 (potasyum tuzu); CTK0H4961; KS-00000WZB; 4696-54-2 (baryum tuzu); 6700-85-2 (kobalt tuzu); 2191-10-8 (kadmiyum tuzu); 5972-76-9 (amonyum tuzu); 6028-57-5 (alüminyum tuzu); 6107-56-8 (kalsiyum tuzu); MolPort-001-792-057; HMS2270A23; Oktanoik asit, analitik standart; 16577-52-9 (lityum tuzu); MGK-5024; ZINC1530416; 1984-06-1 (hidroklorür tuzu); EINECS 242-197-8; kaprilik asit; kapilik asit; kaplik asit; kalik asit; Octoic acid; Octylic acid; acide octanoique; acido octanoico; acidum octanocium; C-8; caprilyc acid; caprylic acid; N-caprylic acid; caprylic acid (natural); caprylic acid (octanoic), natural; caprylic acid natural; caprylic acid synthetic; caprylic acid, synthetic FCC; caprylsaeure; enantic acid; 1-heptane carboxylic acid; 1-heptanecarboxylic acid; hexacid 898; kaprylsaeure; kortacid 0899; lunac 8-98; neo-fat 8; octanoic acid;1-octanoic acid; N-octanoic acid; nat.octanoic acid; octanoic acid (caprylic acid) natural; octanoic acid (natural); octanoic acid naturel; N-octic acid; octoic acid; N-octoic acid; octylic acid; N-octylic acid; prifac 2901;Oktanoik asit; Kaprilik asit; N-oktanoik asit; Oktilik asit; N-kaprilik asit; N-oktilik asit; Kaprilik asit; Kaprilik asit; 14C-etiketli; Kaprilik asit; alüminyum tuzu; Kaprilik asit; amonyak tuzu; Kaprilik asit; baryum tuzu; Kaprilik asit; kadmiyum tuzu; Kaprilik asit; kalsiyum tuzu; Kaprilik asit; sezyum tuzu; Kaprilik asit; kaprilik asit, iridum (+3) tuzu; kaprilik asit, demir (+3) tuzu; kaprilik asit, lantan (+3) tuzu, kaprilik asit, kurşun (+2) tuzu; kaprilik asit, lityum tuzu; kaprilik asit, manganez tuzu; kaprilik asit, nikel (+2) tuzu; kaprilik asit, potasyum tuzu; kaprilik asit, rutenyum (+3) tuzu; kaprilik asit, sodyum tuzu; kaprilik asit, sodyum tuzu, 11C-işaretli; kaprilik asit, kalay tuzu; kaprilik asit, kalay (+2) tuzu; kaprilik asit, çinko tuzu; kaprilik asit, zirkonyum tuzu; kaprilik asit, zirkonyum (+4) tuzu; oktanoat; oktanoik asit; sodyum kaprilat sodyum oktanoat; kaprilikasit; Kaprilik asit; kapirilik asit; caprylicasit; kaprılık asıt; caprylıc acıd, caprylic acid; 14C etiketli kaprilik asit; kaprilik asit, alüminyum tuzu; kaprilik asit, amonyak tuzu; kaprilik asit, baryum tuzu; kaprilik asit, kadmiyum tuzu; kaprilik asit, kalsiyum tuzu; kaprilik asit, sezyum tuzu; kaprilik asit, krom (+2) tuzu; kaprilik asit, kobalt tuzu; kaprilik asit, bakır tuzu; kaprilik asit, bakır (+2) tuzu; kaprilik asit, iridum (+3) tuzu; kaprilik asit, demir (+3) tuzu; kaprilik asit, lantan (+3) tuzu; kaprilik asit, kurşun (+2) tuzu; kaprilik asit, lityum tuzu; kaprilik asit, manganez tuzu; kaprilik asit, nikel (+2) tuzu; kaprilik asit, potasyum tuzu; kaprilik asit, rutenyum (+3) tuzu; kaprilik asit, sodyum tuzu; kaprilik asit, sodyum tuzu, 11C-işaretli; kaprilik asit, kalay tuzu; kaprilik asit, kalay (+2) tuzu; kaprilik asit, çinko tuzu; kaprilik asit, zirkonyum tuzu; kaprilik asit, zirkonyum (+4) tuzu; lityum oktanoat; oktanoat; oktanoik asit; sodyum kaprilat; sodyum oktanoat; oktanoik asit; Kaprilik asit; 124-07-2; n-oktanoik asit; n-kaprilik asit; Oktilik asit; oktoik asit; n-oktilik asit; n-Oktoik asit; neo-yağ 8; 1-heptankarboksilik asit; Enantik asit; Oktik asit; C-8 asit; Caprylsaeure; capryloate; oktanoik asit; Kaprylsaeure; Heksasit 898; Acido oktanoico; 0-kotanoik asit; Asit oktanoique; Acidum octanocium; Kaprilik asit (doğal); Yağ asitleri, C6-10; Kyselina kaprylova; oktilat; Octansaeure; 1-oktanoik asit; Asit oktanoique [Fransızca]; Acido oktanoico [İspanyolca]; Acidum octanocium [Latin]; Kyselina kaprylova [Çek]; NSC 5024; Oktanoik asit [USAN: INN]; LXVIII-OBL58JN025; FEMA No. 2799; CCRIS 4689; HSDB 821; C8: 0; Zımpara 657; Prifac 2901; Lunac 8-95; EINECS 204-677-5; EKTANOİK ASİT (KAPSİTİK ASİT); BRN 1747180; CH3- [CH2] 6-COOH AI3-04162; kaprilik asit, çinko tuzu; OBL58JN025; Chebi: 28.837; kaprilik asit, baryum tuzu; kaprilik asit, sodyum tuzu; NSC5024; WWZKQHOCKIZLMA-UHFFFAOYSA = N; kaprilik asit, kadmiyum tuzu; caprilate; Kaprilik asit, potasyum tuzu; n-kaprilat; kaprilik asit, kalay (+2) tuzu; MFCD00004429; n-oktoat; n-oktilat; FA (8: 0); kaprilik asit, bakır (+2) tuzu; Oktanoik asit,% 99; NCGC00090957-01; C8H16O2; 1-heptanecarboxylate; DSSTox_CID_1645; DSSTox_RID_76259; DSSTox_GSID_21645; OCA; 124-07-2 (Ebeveyn); CAS-124-07-2; CH3- [CH2] 6-COO (-); kaprilik asit, kalay tuzu; kaprilik asit, sezyum tuzu; kaprilik asit, kobalt tuzu; kaprilik asit, bakır tuzu; kaprilik asit, amonyak tuzu; kaprilik asit, kalsiyum tuzu; 14C etiketli kaprilik asit; kaprilik asit, alüminyum tuzu; kaprilik asit, manganez tuzu; kaprilik asit, zirkonyum tuzu; oktanik asit; Kaprilik asit; kaprilik asit, demir (+3) tuzu; kaprilik asit, kurşun (+2) tuzu; asitum oktanoikum; oktanoat kökü; kaprilik asit, iridum (+3) tuzu; kaprilik asit, nikel (+2) tuzu; kaprilik asit, krom (+2) tuzu; kaprilik asit, lantan (+3) tuzu; kaprilik asit, rutenyum (+3) tuzu; (+)-pentacycloanammoxic acid; (3S,4S)-3-hydroxy-4-methyloctanoic acid; 1,2-dioctanoyl-3-ß-D-galactosyl-sn-glycerol; 1-octadecanoyl-2-octanoyl-sn-glycero-3-phosphocholine ; 2-(pentylsulfanyl)acetic acid; 2-hydroxyoctanoic acid; 4,7-dioxooctanoic acid; octanoic acid; caprylic acid; n-octanoic acid; Octylic acid; n-caprylic acid; n-octylic acid; n-Octoic acid

From Wikipedia, the free encyclopedia Jump to navigationJump to search N-(2-Hydroxypropyl) methacrylamide HPMA.png Names IUPAC name N-(2 Hydroxypropyl)methacrylamide Identifiers CAS Number 21442-01-3 ☑ 3D model (JSmol) Interactive image ChemSpider 35398 PubChem CID 38622 InChI SMILES Properties Chemical formula C7H13NO2 Molar mass 143.186 g·mol-1 Appearance White odorless crystals[1] Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa). Infobox references N-(2-Hydroxypropyl)methacrylamide or HPMA is the monomer used to make the polymer poly(N-(2-hydroxypropyl)methacrylamide). Poly(N-(2-hydroxypropyl) methacrylamide); poly(HPMA); pHPMA; PHPMA The polymer is water-soluble (highly hydrophilic), non-immunogenic and non-toxic, and resides in the blood circulation well. Thus, it is frequently used as macromolecular carrier for low molecular weight drugs (especially anti-cancer chemotherapeutic agents) to enhance therapeutic efficacy and limit side effects.[2] Poly(HPMA)-drug conjugate preferably accumulates in tumor tissues via the passive-targeting process (or so-called EPR effect). Due to its favorable characteristics, HPMA polymers and copolymers are also commonly used to produce synthetic biocompatible medical materials such as hydrogels. The development of pHPMA as anti-cancer drug delivery vehicles is initiated by Dr. Jindřich Kopeček and colleagues at the Czech (-oslovak) Academy of Sciences in Prague in the mid-1970s.[3] Prior to this, it was used as a plasma expander. The Kopeček Laboratory designed and developed HPMA copolymer-drug conjugates as a lysosomal delivery vehicle to cancer cells. The concept of using pHPMA as polymeric drug carriers has opened a new perspective in modern pharmaceutical science, and developed into the first polymer-drug conjugate entering clinical trials (i.e. PK1; HPMA copolymer-doxorubicin conjugate).[4] The HPMA copolymers are also used as a scaffold for iBodies, polymer-based antibody mimetics. Hydrolyzed Polymaleic Anhydride (HPMA) CAS No. 26099-09-2 Structural Formula: Hydrolyzed Polymaleic Anhydride (HPMA) Properties: HPMA is a low molecular weight polymeride, with average molecular weight 400-800. No toxicity, soluble in water, high chemical and thermal stability, decomposed temperature above 330℃. HPMA has obvious threshold effect under high temperature (350℃) and high pH(8.3)level, suitable to be used in alkaline water system or built with agents. It has good scale inhibition against carbonate and phosphate scales under temperature 300℃ with effective time as long as 100 hours. Due to its good scale inhibition and high temperature tolerance properties, HPMA is widely used in desalination plant of flash vaporization equipment, low pressure boiler, steam locomotive, crude oil evaporation, petroleum pipeline, and industrial circulating cool water systems. In addition, HPMA has good corrosion inhibition effect when used together with zinc salt. HPMA can also used as additives for cement. Specification: Items Index Appearance Pale yellow to umber transparent liquid Solid content % 50.0 min Bromine value mg/g 50.0 max pH(1% water solution) 2.0-3.0 Density (20℃) g/cm3 1.22-1.25 Usage: HPMA is usually used together with organic phosphonate at dosage of 1-15ppm for circulating cool water system, oilfield fill water, crude oil dewatering and low-pressure boilers. HPMA has good scale inhibition (98%) and scale stripping properties. When used together with zinc salts, it can effectively inhibit carbon steel corrosion. Package and Storage: 200L plastic drum, IBC(1000L), customers' requirement. storage for one year in shady room and dry place. Safety Protection: Acidity, Avoid contact with eye and skin, once contacted, flush with water. Jindřich Kopeček* and Pavla Kopečková Author information Copyright and License information Disclaimer The publisher's final edited version of this article is available at Adv Drug Deliv Rev See other articles in PMC that cite the published article. Go to: Abstract The overview covers the discovery of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, initial studies on their synthesis, evaluation of biological properties, and explorations of their potential as carriers of biologically active compounds in general and anticancer drugs in particular. The focus is on the research in the authors' laboratory - the development of macromolecular therapeutics for the treatment of cancer and musculoskeletal diseases. In addition, the evaluation of HPMA (co)polymers as building blocks of mod and new biomaterials is presented: the utilization of semitelechelic poly(HPMA) and HPMA copolymers for the modification of biomaterial and protein surfaces and the design of hybrid block and graft HPMA copolymers that self-assemble into smart hydrogels. Finally, suggestions for the design of second-generation macromolecular therapeutics are portrayed. Keywords: N-(2-hydroxypropyl)methacrylamide, Cancer, Cleavable spacers, Bioconjugates, Hydrogels, Self-assembly Go to:

1. Introduction

Water-soluble polymers have a distinguished record of clinical relevance. They have been used in the clinics and/or clinical trials for the modification of proteins, modification of liposomes, surface modification of biomaterials, and as carriers of drugs, genes, and oligonucleotides. The concept of water-soluble macromolecular carriers of (anticancer) drugs has evolved continuously over the last century. In 1906, Ehrlich coined the phrase "magic bullet", and recognized the importance of biorecognition for successful drug delivery [1]. DeDuve discovered the lysosomotropism of macromolecules and the high enzymatic activity localized in the lysosomal compartment [2]. The conjugation of drugs to synthetic and natural macromolecules was initiated more than 50 years ago. Jatzkewitz used a dipeptide spacer to attach a drug (mescaline) to polyvinylpyrrolidone in the early fifties [3]. Ushakov's group in St. Petersburg (Leningrad) synthesized numerous water-soluble polymer-drug conjugates in the sixties and seventies [4-6]. Mathé et al. pioneered conjugation of drugs to immunoglobulins, setting the stage for targeted delivery [7]. Ringsdorf presented a clear summary of the field in 1975 [8]. The research in our laboratory, first in Prague, later in Utah, concentrated on hydrophilic polymers as biomaterials and drug carriers. The early program in Prague on the biocompatibility of hydrophilic polymers resulted in clinical application of hydrogels as implants [9] and in the design of water-soluble polymers as carriers of biologically active compounds (drugs) [10]. This special volume is devoted to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. It is an opportunity to review what was done and identify directions for future research. The HPMA development and data presented will be related mostly to the authors' laboratory, not to overlap with other author's contributions in this volume. The work done with HPMA copolymers as drug carriers, protein, and surface modifiers, and as synthetic components in smart hybrid biomaterials design has been summarized. More details and work from other laboratories may be found in the other chapters in this volume that cover more focused topics.

Go to: 2. Origins 2.1. Early history Our research in the sixties was concentrated on the hydrophilic biomedical polymers based on hydrophilic esters and N-substituted amides of (meth)acrylic acid. The focus was on four tasks [10]: a) selection of a suitable group of polymers and the study of their chemical properties - polymerization kinetics, stability, mechanical properties, permeability, possibility of introduction of functional (reactive) groups; b) study of the relationship between the chemical and physical structure of crosslinked polymers (hydrogels) and their biocompatibility; c) study of the interaction of water-soluble polymers with living organisms - using evaluation criteria from blood plasma expanders; and d) modification of soluble polymers by attachment of biologically active compounds (drugs, hormones, enzymes) as well as the modification of the polymer backbone and side-chains with enzymatically degradable sequences. The choice of HPMA for development as drug carrier was not random. Based on the detailed studies of the relationship between the structure of hydrophilic polymers and their biocompatibility [11-21], we have chosen N-substituted methacrylamides as our target because the α-carbon substitution and the N-substituted amide bond ensured hydrolytic stability of the side-chains. We synthesized a series of compounds trying to identify a crystalline monomer for easy purification and reproducible synthesis. The first crystalline N-substituted methacrylamide we succeeded to synthesize, HPMA, was chosen for future development [22,23].

2.2. First HPMA copolymer drug and/or protein conjugates

The research on the use of HPMA copolymers as drug carriers commenced in the early 70s. In April 1974 we filed two patent applications [24,25] which covered the synthesis of N-substituted (meth)acrylamides containing oligopeptide sequences and their application as drug (and other biologically active compounds) carriers.The first HPMA copolymer-drug (N-(4-aminobenzensulfonyl)-N′-butylurea) conjugate (Fig. 1) was presented at the Prague Microsymposiumon Polymers in Medicine in 1977 [26] and published [27]. At the same time studies in the modification of proteins were initiated - HPMA copolymer-insulin [28] and HPMA copolymer-chymotrypsin [29,30] conjugates. An external file that holds a picture, illustration, etc. Object name is nihms159442f1.jpg Fig. 1 First HPMA copolymer-drug conjugate; HPMA copolymer-N-(4-aminobenzenesulfonyl)- N′-butylurea [26,27].

Two major synthetic routes were used for the synthesis of HPMA copolymer-drug conjugates, copolymerization and polymer-analogous attachment [10,21]. Copolymerization of HPMA with a polymerizable derivative of a drug, e.g., N-(4-aminobenzensulfonyl)-N′-butylurea [27] is the example of the first route. Insulin [28], chymotrypsin [29,30] and ampicillin [31] (Fig. 2) were attached to HPMA copolymers by aminolysis [32,33] of reactive polymeric precursors. The development of HPMA copolymers is summarized in Table 1. An external file that holds a picture, illustration, etc. Object name is nihms159442f2.jpg Fig. 2 HPMA copolymer-ampicillin conjugate [31]. Table 1 Milestones in HPMA (co)polymer research. Year Study Publication 1973 First synthesis J. Kopeček, H. Bažilová, Poly[N-(2-Hydroxypropyl)methacrylamide]. 1. Radical polymerization and copolymerization. Europ. Polym. J. 9 (1973) 7-14. 1974 Characterization of PHPMA solution properties M. Bohdanecký, H. Bažilová, J. Kopeček, Poly[N-(2-Hydroxypropyl)methacrylamide]. II. Hydrodynamic properties diluted polymer solutions. Europ. Polym. J. 10 (1974) 405-410. 1974 First hydrogels J. Kopeček, H. Bažilová, Poly[N-(2-Hydroxypropyl)methacrylamide]. III. Crosslinking copolymerization. Europ. Polym. J. 10 (1974) 465-470. 1976 First enzymatic release of ligand from a polymer conjugate in vitro J. Drobník, J. Kopeček, J. Labský, P. Rejmanová, J. Exner, V. Saudek, J. Kálal, Enzymatic cleavage of side-chains synthetic water-soluble polymers. Makromol. Chem. 177 (1976) 2833-2848. 1978 First HPMA modified protein V. Chytrý, A. Vrána, J. Kopeček, Synthesis and activity of a polymer which contains insulin covalently bound on a copolymer of N-(2-hydroxypropyl)methacrylamide and N-methacryloylglycylglycine 4-nitrophenyl ester. Makromol. Chem. 179 (1978) 329-336. 1979 First HPMA-drug conjugate B. Obereigner, M. Burešová, A. Vrána, J. Kopeček, Preparation of polymerizable derivatives of N-(4-aminobenzenesulfonyl)-N′-butylurea. J. Polym. Sci. Polym. Symp. 66 (1979) 41-52. 1981 First enzymatic release of ligand from a polymeric substrate by a polymer-modified enzyme J. Kopeček, P. Rejmanová, V. Chytrý, Polymers containing enzymatically degradable bonds 1. Chymotrypsincatalyzed hydrolysis of p-nitroanilides of phenylalanine and tyrosine attached to side-chains of copolymers of N-(2-hydroxypropyl)methacrylamide. Makromol. Chem. 182 (1981) 799-809. 1981 First enzymatic release of ligand from a polymer conjugate in vivo J. Kopeček, I. Cífková, P. Rejmanová, J. Strohalm, B. Obereigner, K. Ulbrich, Polymers containing enzymatically degradable bonds. 4. Preliminary experiments in vivo. Makromol. Chem. 182 (1981) 2941-2949.

1982 First degradable hydrogels K. Ulbrich, J. Strohalm, J. Kopeček, Polymers containing enzymatically degradable bonds. VI. Hydrophilic gels cleavable by chymotrypsin. Biomaterials 3 (1982) 150-154. 1985 First HPMA-drug-antibody conjugate B. Říhová, J. Kopeček, Biological Properties of targetable poly[N-(2-hydroxypropy)methacrylamide]-antibody conjugates. J. Controlled Release 2 (1985) 289-310. 1994 First combination therapy using polymer-bound drugs N.L. Krinick, Y. Sun, D. Jonyer, J.D. Spikes, R.C. Straight, J. Kopeček, A polymeric drug delivery system for the simultaneous delivery of drugs activatable by enzymes and/or light. J. Biomat. Sci. Polym. Ed. 5 (1994) 211-222. 1995 First semitelechelic HPMA S. Kamei, J. Kopeček, Prolonged blood circulation in rats of nanospheres surface-modified with semitelechelic [N-(2-hydroxypropyl)methacrylamide]. Pharmaceutical Res. 12 (1995) 663-668. 1999 First clinical trials P.A. Vasey, S.B. Kaye, R. Morrison, C. Twelves, P. Wilson, R. Duncan, A.H. Thomson, L.S. Murray, T.E. Hilditch, T. Murray, S. Burtles, D. Fraier, E. Frigerio, J. Cassidy, and on behalf of the Cancer Research Campaign Phase I/II Committee, Phase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: first member of a new class of chemotherapeutic agents-drug-polymer conjugates. Clin. Cancer Res. (1999) 83-94. 1999 First ATRP polymerization M. Teodorescu, K. Matyjaszewski, Atom transfer radical polymerization of (meth)acrylamides. Macromolecules (1999) 4826-4831. 1999 First self-assembly into hybrid hydrogels C. Wang, R.J. Stewart, J. Kopeček, Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains. Nature 397 (1999) 417-420. 2005 First RAFT polymerization C.W. Scales, Y.A. Vasilieva, A.J. Convertine, A.B. Lowe, C.L. McCormick, Direct, controlled synthesis of the nonimmunogenic, hydrophilic polymer, poly(N-(2-hydroxypropyl)methacrylamide) via RAFT in aqueous media. Biomacromolecules 6 (2005) 1846-1850.

2009 First self-assembly at cell surface K. Wu, J. Liu, R.N. Johnson, J. Yang, J. Kopeček, Drug-free macromolecular therapeutics: induction of apoptosis coiled-coil mediated crosslinking of antigens at cell surface. Submitted Open in a separate window

2.3. Development of oligopeptide spacers

Macromolecules are internalized by cells via endocytosis and ultimately localize in the (enzyme rich) lysosomal compartment. Consequently, we developed HPMA copolymers containing enzymatically degradable bonds (Fig. 3) [34]. Oligopeptide side-chains were designed as drug attachment/release sites [35] and shown to be degradable in vivo [36]. An external file that holds a picture, illustration, etc. Object name is nihms159442f3.jpg Open in a separate window Fig. 3 HPMA copolymers containing enzymatically cleavable bonds [30,34,37-45,47-49,55].

The relationship between the structure of oligopeptide sequences and the rate of enzymatically catalyzed release of a drug or drug model was studied thoroughly. First, model enzymes, chymotrypsin [30,37-39], trypsin [40], and papain [41] were evaluated, followed by intracellular (lysosomal) enzymes. A study with a mixture of lysosomal enzymes revealed that cysteine (thiol) proteinases were responsible for the cleavage of these polymer-drug conjugates [42,43]. Subsequently, polymers were designed to match the specificity of individual cysteine proteinases: cathepsins B [44], L, H, and artificial mixtures of lysosomal enzymes [45]. The stability of the oligopeptide side-chains in blood plasma and serum was verified [46]. Based on these results it was possible to control the degradability of HPMA copolymers by a particular enzyme. Early in vivo validation of the design was achieved - the drug model (p-nitroaniline) was released in in vivo following intravenous (i.v.) administration in Wistar rats [36].

Later, oligopeptide spacers were combined with self-immolative groups. For bone-specific delivery of prostaglandins a cathepsin K sensitive spacer combined with a 1,6-elimination unit was designed [50]; for oral delivery of 9-aminocamptothecin a combination of a reducible aromatic azo bond with a 1,6-elimination unit was employed [51].

The first degradable polymer carriers based on HPMA were also reported at the Polymers in Medicine Microsymposium in the Prague in 1977 [52] and at conferences in Varna [53] and Tashkent [54]. We used the oxidized insulin B chain (it contains two amino groups at positions 1 and 29) to prepare branched, water-soluble HPMA copolymers by reacting insulin B-chain with HPMA copolymers containing side-chains terminated in p-nitrophenyl esters. The polymers were cleavable (Fig. 4), so we chose the sequence 23-25 (Gly-Phe-Phe) from the insulin B-chain (the bond originating at amino acid 25 is cleavable by chymotrypsin) and synthesized branched, soluble high molecular weight enzymatically degradable copolymers containing the Gly-Phe-Phe segments in crosslinks connecting primary chains [38]. The latter type of polymer carrier was evaluated in vivo in rats and it was shown that the branched polymer carrier is degradable and its molecular weight distribution decreases with time following i.v. administration [36]. These experiments demonstrated the possibility to manipulate the intravascular half-life of polymeric carriers based on HPMA.

An external file that holds a picture, illustration, etc. Object name is nihms159442f4.jpg Fig. 4 Branched HPMA copolymers containing the GFF degradable sequence in crosslinks; this sequence mimics the amino acid residues 23-25 of the insulin B chain [38,52]. 2.4. Validation of the targetability of HPMA copolymer-drug conjugates The choice and design of a targeting system has to be based on a sound biological rationale. The design of the first targetable HPMA copolymer was based on the observation [56] that small changes in the structure of glycoproteins lead to dramatic changes in the fate of the modified glycoprotein in the organism. When a glycoprotein (ceruloplasmin) was administered into rats, a long intravascular half-life was observed. However, when the terminal sialic acid was removed from ceruloplasmin, the asialoglycoprotein (asialoceruloplasmin) formed contains side-chains exposing the penultimate galactose units. The intravascular half-life of the latter was dramatically shortened due to the biorecognition of the molecule by the asialoglycoprotein receptor on the hepatocytes. This receptor recognizes galactose and N-acetylgalactosamine moieties [56]. To determine if one can mimic this process with a synthetic macromolecule, we synthesized HPMA copolymers with N-methacryloylglycylglycine p-nitrophenyl ester and attached galactosamine by aminolysis [57]. These copolymers behaved similarly to the glycoproteins and were biorecognizable in vivo (Fig. 5). Their clearance from the bloodstream was related to the N-acylated galactosamine content (1-11 mol%) of the HPMA copolymer [57-59]. Separation of the rat liver into hepatocytes and non-parenchymal cells indicated that the polymer is largely associated with hepatocytes, and density-gradient subcellular fractionation of the liver confirmed that the HPMA copolymers were internalized by liver cells and transported, with time, into the secondary lysosomes [59,60]. It was very important to find that HPMA copolymers containing side-chains terminated in galactosamine and anticancer drug adriamycin also preferentially accumulated in the liver, i.e., it appeared that non-specific hydrophobic interactions with cell membranes did not interfere with the biorecognition by hepatocytes [61]. An external file that holds a picture, illustration, etc. Object name is nihms159442f5.jpg Open in a separate window Fig. 5 Validations of the targetability of HPMA copolymers. N-acylated galactosamine as the targeting moiety was chosen to mimic the glycoprotein-asialoglycoprotein system [57-59]. In parallel, efforts on the targetability of HPMA copolymer-antibody conjugates started. First HPMA copolymer conjugates with polyclonal and monoclonal anti-Thy-1.2 antibodies and anti-FITC (fluorescein isothiocyanate) antibodies were evaluated. Targetable conjugates containing daunomycin were synthesized and in vitro experiments have shown two orders of magnitude enhanced cytotoxicity of the targeted conjugate (when compared to the nontargeted one) [62]. The targetability and activity of anti-Thy1.2 conjugates with HPMA copolymer-daunomycin conjugates was proven in vivo on a mouse model [63]. Anti-Thy1.2 antibodies were also efficient in targeting HPMA copolymer-photosensitizer (chlorin e6) conjugates [64].

2.5. Early interdisciplinary collaborations

At the beginning of the eighties, we started collaborations with coworkers from the biological field: John Lloyd and Ruth Duncan from the University of Keele in United Kingdom, and Blanka Říhová from the Institute of Microbiology in Prague. The collaboration with the Keele group was initiated by Helmuth Ringsdorf who gave a lecture at the 1977 Prague symposium (where Kopecek presented first HPMA copolymer-drug conjugates and biodegradable carriers based on HPMA). After the meeting Ringsdorf suggested to Lloyd to contact Kopecek because he thought that the collaboration would be beneficial for both. Kopecek met Lloyd in Dresden in July 1978 and they agreed on the evaluation of HPMA copolymer conjugates. First samples were synthesized (different side-chains terminated in p-nitroanilide as drug model) and evaluated at Keele for their cleavability by lysosomal enzymes [42,65] and their stability in blood plasma and serum [46]. More than 300 different polymer structures containing oligopeptide sequences were synthesized in the Prague laboratory [24,25,35,47], and biological properties of a number of them evaluated at Keele within a 10 year period [66,67]. The collaboration with Vladimír Kostka and coworkers from the Institute of Organic Chemistry and Biochemistry in Prague on the cleavability of peptide sequences in HPMA copolymers by cathepsin B [44, Fig. 4], the most important lysosomal cysteine proteinase, resulted in the identification of GFLG sequence, which is incorporated in all conjugates used in clinical trials. From the two fastest cleaving oligopeptides, GFLG and GFTA (see Fig. 3, example 5), we have chosen the GFLG sequence over the GFTA to avoid T; at that time we were worried about the potential immunogenicity. In 1978 Kopecek gave a lecture at the Institute of Microbiology in Prague. After the lecture he discussed with Říhová and the collaboration with her group on the immunogenicity/biocompatibility [69-72] and biorecognition (targeting) [62-64] of HPMA conjugates commenced. These collaborations resulted in the filing of "Polymeric drugs" patent application in 1985 [68]. Kopecek coined the name for the HPMA copolymers evaluated in clinical trials as PK1 and PK2(P for Prague, K for Keele) (Fig. 6). An external file that holds a picture, illustration, etc Object name is nihms159442f6.jpg

Fig. 6

Structures of PK1 and PK2, first HPMA copolymers evaluated in clinical trials [68]. Conjugate PK1 contains doxorubicin bound to HPMA copolymer via a tetrapeptide sequence stable in the blood stream but susceptible to enzymatically catalyzed hydrolysis in the lysosomes. Conjugate PK2 contains in addition side-chains terminated in N-acylated galactosamine complementary to the asialoglycoprotein receptor on hepatocytes.

3. HPMA copolymer-drug conjugates

The early experiments provided the foundation for the development of HPMA copolymers as drug carriers. As in the majority of new scientific areas, the research initially focused on the accumulation of basic data on the structure-properties relationship. The summary of research in areas we consider important for the development of clinically relevant HPMA copolymer conjugates follows:

Rationale

HPMA copolymer-drug conjugates are nanosized (5-20 nm) water-soluble constructs. Their unique structural, physicochemical, and biological properties are advantageous when compared to low molecular weight drugs. The concept of targeted polymer-drug conjugates was developed to address the lack of specificity of low molecular weight drugs for cancer cells. This approach was based on the work of DeDuve, who realized that the endocytic pathway is suitable for lysosomotropic drug delivery [2]. The features needed to design an effective conjugate [10,21,35,73-75] comprise a polymer-drug linker that is stable during transport [46] and able to release the drug in the lysosomal compartment of the target cell at a predetermined rate [34,47,75], adequate physicochemical properties of the conjugate (solubility, conformation in the biological environment) [48,76-78], and the capability to target the diseased cell or tissue by an active (receptor-ligand) [75] or a passive (pathophysiological) mechanism [79]. Since the activity of many drugs depends on their subcellular location, a mechanism for the manipulation of the subcellular drug location would be beneficial.

 

The advantages of polymer-bound drugs (when compared to low molecular weight drugs) are (reviewed in [10,47,67,73-75,80,81]): a) active uptake by fluid-phase pinocytosis (non-targeted polymer-bound drug) or receptor-mediated endocytosis (targeted polymer-bound drug), b) increased active accumulation of the drug at the tumor site by targeting [82-84], c) increased passive accumulation of the drug at the tumor site by the enhanced permeability and retention (EPR) effect [85], d) long-lasting circulation in the bloodstream [86], e) decreased non-specific toxicity of the conjugated drug [82], f) decreased immunogenicity of the targeting moiety [82], f) immunoprotecting and immunomobilizing activities [87], and g) modulation of the cell signaling and apoptotic pathways [88-94].

3.1. Enhanced permeability and retention effect

The enhanced permeability and retention (EPR) effect is the predominant mechanism by which soluble macromolecular anticancer drugs exhibit their therapeutic effect on solid tumors. The phenomenon is attributed to high vascular density of the tumor, increased permeability of tumor vessels, defective tumor vasculature, and malfunctioning or suppressed lymphatic drainage in the tumor interstitium [79]. Other factors, however, may have an opposite effect. For example, a high interstitial pressure may result in a convective fluid flow from the center of the tumor to the periphery, which might carry macromolecules [95]. Nevertheless, a number of studies showed increased accumulation of macromolecules in tumors as compared to that in normal tissue [85,96]. The degree of accumulation was dependent on molecular weight [85,86], charge [97,98], and their overall hydrophobic -hydrophilic character. The tumor type and microenvironment may influence its transport characteristics (pore cutoff size) [99].

The efficiency of extravasation into solid tumors depends on the concentration gradient between the vasculature and tumor tissue and time. Consequently, high molecular weight (long-circulating) polymer conjugates accumulate efficiently in tumor tissue [85] due to the EPR effect [79,100]. However, if they possess a non-degradable backbone, they may deposit and accumulate in various organs [18]. We have previously synthesized high molecular weight carriers by connecting HPMA chains via lysosomally degradable oligopeptide sequences [34] to form water-soluble branched conjugates [36,38-41,101-103]. Following intravenous (i.v.) administration to rats, the oligopeptide crosslinks were cleaved and the resulting lower molecular weight polymer chains were excreted into the urine [36]. These water-soluble copolymers were synthesized by crosslinking (short of gel point) of HPMA copolymer precursors (containing oligopeptide side-chains terminated in a reactive ester group) with diamines.

Later, we designed a new, reproducible synthetic pathway for long-circulating HPMA copolymers [85,104]. New crosslinking agents were synthesized and high molecular weight copolymers prepared by crosslinking copolymerization. The composition of the monomer mixture, however, has to be such that at the end of the polymerization the system is short of the gel point (water-soluble). This method [104] is also suitable for the synthesis of HPMA copolymers, which contain, in addition to oligopeptide crosslinks, oligopeptide side-chains terminated in doxorubicin (DOX) (or other anticancer drugs).

 

The influence of the molecular weight of such conjugates on their biological activity was evaluated [85]. Copolymerization of HPMA, a polymerizable derivative of DOX (N-methacryloylglycylphenylalanylleucylglycyl doxorubicin) and a crosslinking agent, N2,N5-bis(N-methacryloylglycylphenylalanylleucylglycyl) ornithine resulted in high molecular weight, branched, water-soluble HPMA copolymers containing lysosomally degradable oligopeptide sequences in the crosslinks as well as in side-chains terminated in DOX. Four conjugates with Mw of 22, 160, 895, 1230 kDa were prepared. Biodistribution of the conjugates and their treatment efficacy in nu/nu mice bearing s.c. human ovarian OVCAR-3 carcinoma xenografts were determined (Fig. 7). The half-life of conjugates in the blood was up to 5 times longer and the elimination rate from the tumor was up to 25 times slower as the Mw of conjugates increased from 22 to 1230 kDa. The treatment with HPMA copolymer-bound DOX possessing an Mw higher than 160 kDa inhibited the tumor growth more efficiently than that of 22 kDa or free DOX(p<0.02). The data clearly indicated that the higher the molecular weight of the conjugate the higher the treatment efficacy of human ovarian xenografts in nu/nu mice [85].

An external file that holds a picture, illustration, etc. Object name is nihms159442f7.jpg Open in a separate window Fig. 7 Long-circulating HPMA copolymer-DOX (P-DOX) conjugates of different molecular weight (Mw). (A) Chemical structure of HPMA copolymer-doxorubicin conjugate containing glycylphenylalanylleucylglycine side-chains and N2,N5-bis(N-methacryloylglycylphenylalanylleucylglycyl)ornithine crosslinker [104]; (B) concentration of DOX in OVCAR-3 carcinoma xenografts in nu/nu mice after i.v. bolus of free DOX or P-DOX of different Mw; (C) growth inhibition of s.c. human ovarian OVCAR-3 carcinoma xenografts in nu/nu mice by long-circulating P-DOX conjugates. The mice received i.v. injection of 2.2 mg/kg DOX equivalent dose as P-DOX of different Mw [85].

3.2. Multidrug resistance

The acquired resistance of malignant tumors to chemotherapeutic agents remains the major cause of cancer therapy failure [105]. A membrane glycoprotein, termed P-glycoprotein, has been shown to be responsible for cross-resistance to a broad range of structurally and functionally distinct cytotoxic agents. This glycoprotein, encoded in humans by the mdr1 gene, functions as an energy-dependent efflux pump and removes cytotoxic agents from the resistant cells. The elucidation of the function of P-glycoprotein [106], other ATP-driven efflux pumps [107], as well as other mechanisms of multidrug resistance [108] has had a major impact on the understanding of multidrug resistance in human tumors. The exclusion of the polymer-drug conjugate from the cytoplasmof the cell, through intracellular trafficking in membrane-limited organelles, renders the efflux pumps ineffective. Furthermore, subcellular trafficking along the endocytic pathway from the plasmamembrane to the perinuclear region changes the gradient of distribution of drugs inside cells [109,110]. The concentration gradient of free drugs is directed from the plasma membrane to the perinuclear region, in contrast to polymer-bound drugs, which have a gradient in exactly the opposite direction. The drug, released from the polymeric carrier in the lysosomal compartment, enters the cytoplasm in the perinuclear region. Consequently, the probability of its interaction with nuclear DNA and/or topoisomerase II is higher than the probability of its recognition by the P-glycoprotein efflux pump [109].

We hypothesized that HPMA copolymer-bound DOX [P(GFLG)- DOX] (P is the HPMA copolymer backbone) would behave differently than free DOX during long term incubation with cancer cells. To verify the hypothesis, we have studied the effect of free DOX and P(GFLG)- DOX on the induction of multidrug resistance and changes in metabolism in human ovarian carcinoma A2780 cells during repeated cyclic (chronic) exposure [111]. Such experiments are of therapeutic relevance. The development of multidrug resistance during adaptation of sensitive human ovarian carcinoma A2780 cells to free DOX and P(GFLG)-DOX was analyzed. Adaptation of sensitive A2780 cells to repeated action of free DOX augmented cellular resistance to DOX and finally led to the over-expression of the MDR1 gene. On the other hand, P(GFLG)-DOX induced neither the multidrug resistance with or without MDR1 gene expression, nor the adaptation of the sensitive A2780 cells to free DOX [111].

The in vivo comparison of the efficiency of DOX and P(GFLG)-DOX in solid tumor mice models of DOX sensitive (A2780) and resistant (A2780/AD) human ovarian carcinoma [89] demonstrated the advantages of polymer conjugation. Free DOX was effective only in sensitive tumors decreasing the tumor size about three times, while P(GFLG)-DOX decreased the tumor size 28 and 18 times in the sensitive and resistant tumors, respectively (Fig. 8). An enhanced accumulation of P(GFLG)- DOX in the tumor was observed, whereas only low concentrations of DOX were detected in other organs (brain, liver, kidney, lung, spleen, and heart) following P(GFLG)-DOX administration [89].

An external file that holds a picture, illustration, etc. Object name is nihms159442f8.jpg Fig. 8 Effect of free DOX (squares) and HPMA copolymer-bound DOX (triangles) on the growth of sensitive A2780 and multidrug resistant A2780/AD human ovarian carcinoma xenografts in female nu/nu mice. Mice were treated i.p. 6 times over 3 weeks (1st and 4th day of each week) with the maximum tolerated dose of free DOX (5 mg/kg) and P(GFLG)- DOX (25 mg/kg). Circles - control tumor. Means±SE are shown [89].

3.3. Combination therapy with polymer-bound drugs

Photodynamic therapy is a newer paradigm in anticancer therapy that involves activation of specific compounds called photosensitizers with specific wavelengths of light to induce cell death. Illumination of these compounds results in the generation of free radicals and singlet oxygen, which cause cell damage and ultimately cell death. A combination of chemotherapy and photodynamic therapy may result in a synergistic response, resulting in a better cure rate than monotherapy. On two cancer models, Neuro 2A neuroblastoma [112] and human ovarian carcinoma heterotransplanted in nude mice [113], we have shown that combination therapy with HPMA copolymer-anticancer drug [DOX and meso chlorin e6 mono(N-2-aminoethylamide) {Mce6}] conjugates showed tumor cures that could not be obtained with either chemotherapy or photodynamic therapy alone. Cooperativity of the action of both drugs contributed to the observed effect [114]. Based on biodistribution data [115], we hypothesized that combination therapy of s.c. human ovarian carcinoma OVCAR-3 xenografts in nude mice using multiple doses/irradiation of P(GFLG)-Mce6 and P(GFLG)-DOX may acquire low effective doses without sacrificing the therapeutic efficacy. Indeed, 10 out of 12 tumors exhibited complete responses in the group of mice receiving multiple photodynamic therapy (PDT) plus multiple chemotherapy [116].

Finally, we have demonstrated the advantages of targeted combination chemotherapy and photodynamic therapy using OV-TL16- targeted HPMA copolymer-DOX and HPMA copolymer-mesochlorin e6 conjugates. OV-TL16 antibodies are complementary to the OA-3 antigen (CD47) present on the majority of ovarian cancers. The immunoconjugates (Fig. 9) preferentially accumulated in human ovarian carcinoma OVCAR-3 xenografts in nude mice with a concomitant increase in therapeutic efficacy when compared with non-targeted conjugates [83]. The targeted conjugates suppressed tumor growth for the entire length of the experiment (>60 days; unpublished data).

An external file that holds a picture, illustration, etc. Object name is nihms159442f9.jpg Open in a separate window Fig. 9 Efficacy of combination chemotherapy and photodynamic therapy of OVCAR-3 xenografts in nude mice with non-targeted and OV-TL16 antibody-targeted HPMA copolymer conjugates. Therapeutic efficacy of combination therapy of HPMA copolymer-bound Mce6 (P(GFLG)-Mce6) and DOX (P(GFLG)-DOX) targeted with OV-TL 16 antibodies toward OVCAR-3 xenografts was compared to non-treated xenografts and non-targeted combination chemotherapy and photodynamic therapy. Equivalent doses of targeted combination therapy enhanced the tumor-suppressive effect as compared to non-targeted combination therapy. Dose administered: 2.2 mg/kg DOX equivalent and 1.5 mg/kg Mce6 equivalent. Irradiation for photodynamic therapy: 650 nm, 200 mW/cm2 18 h after administration [83, unpublished].

The combination index (CI) analysis was used to quantify the synergism, antagonism, and additive effects of binary combinations of free and HPMA copolymer-bound anticancer drugs, 2,5-bis(5-hydroxymethyl- 2-thienyl)furan (SOS), DOX, and mesochlorin e6 mono-ethylenediamine (Mce6) in anticancer effect toward human renal carcinoma A498 cells. The combination of SOS+DOX proved to be synergistic over all cell growth inhibition levels. All other combinations exhibited synergism in a wide range of drug effect levels [117]. Similarly, the targeted (using Fab′ of OV-TL16 antibody) and nontargeted targeted HPMA copolymer-drug conjugates, P(GFLG)-Mce6 and P(GFLG)-SOS, were evaluated against human ovarian carcinoma OVCAR-3 cells. The observations that most combinations produced synergistic effects will be important for clinical translation [118].

In collaboration with Satchi-Fainaro's laboratory at the University of Tel Aviv a new therapeutic strategy for bone neoplasms using combined targeted polymer-bound angiogenesis inhibitors was developed [119]. The aminobisphosphonate alendronate (ALN), and the potent anti-angiogenic agent TNP-470 were conjugated with HPMA copolymer. Using reversible addition-fragmentation chain transfer (RAFT) polymerization, we synthesized a HPMA copolymer-ALN-TNP-470 conjugate bearing a cathepsin K-cleavable linker, a protease overexpressed in bone tissues. Free and conjugated ALNTNP- 470 demonstrated their synergistic anti-angiogenic and antitumor activity by inhibiting proliferation, migration and capillary-like tube formation of endothelial and osteosarcoma cells. The bi-specific HPMA copolymer conjugate reduced vascular hyperpermeability and remarkably inhibited human osteosarcoma growth in mice by 96%. These findings indicate that HPMA copolymer-ALN-TNP-470 is the first narrowly dispersed anti-angiogenic conjugate synthesized by RAFT polymerization that targets both the tumor epithelial and endothelial compartments warranting its use on osteosarcomas and bone metastases (Fig. 10) [119].

Inhibition of MG-63-Ras human osteosarcoma growth in mice by HPMA copolymer-ALN-TNP470 conjugate. (A) Structure of the conjugate; (B) effects of free (open triangles) or conjugated (closed triangles) ALN and TNP-470 on MG-63-Ras human osteosarcoma tumor growth compared to vehicle-treated group (closed squares) and dissected tumors images. Scale bar represents 10 mm. Data represent mean±S.E. (n=5 mice per group). Adapted from [119].

3.4. Novel targeting strategies

As discussed in 3.1, HPMA copolymer-drug conjugates accumulate passively in solid tumors as a result of the (molecular weight dependent) enhanced permeation and retention (EPR) effect [85]. Active targeting of HPMA copolymer-drug conjugates can be achieved with the incorporation of cancer cell-specific ligands, such as carbohydrates, lectins, antibodies, antibody fragments, and peptides, resulting in enhanced uptake of conjugates by cancer cells through receptor-mediated endocytosis with concomitant improvement of therapeutic efficacy [120,121].

Among different cancer targeting molecules, peptides are of particular interest. Enhanced peptide targeting efficiency can be achieved through multivalent interactions [122] between targets and HPMA copolymer-peptide conjugates containing multiple copies of peptides within a single polymer chain (Fig. 11) [123].

Multivalency effect in the biorecognition of HPMA copolymer-peptide-DOX conjugates. Inhibition of Raji B cell growth by exposure to HPMA copolymer-DOX (P (GFLG)-DOX) conjugate containing varying amount of targeting peptide, EDPGFFN-VEIPEF, per macromolecule. (A) Structure of conjugate; (B) inhibition of Raji B cell growth by P(GFLG)-DOX (no targeting peptide), P(GFLG)-DOX containing 1.9 mol% targeting peptide, and P(GFLG)-DOX containing 3.9 mol% targeting peptide. Adapted from [123].

Combinatorial approaches, such as phage display or synthetic peptide libraries, are suitable for the identification of targeting peptides. Overexpression of the CD21 receptor was found on lymphoblastoid cell lines such as Raji cells; consequently, we have used these techniques to identify targeting moieties for lymphomas [124,125]. With phage display, five distinctive peptides (RMWPSSTVNLSAGRR, PNLDFSPTCSFRFGC, GRVPSMFGGHFFFSR, RLAYWCFSGLFLLVC, and PVAAVSFVPYLVKTY) were identified as ligands of CD21 receptor. The dissociation constants of selected peptides were determined to be in the micromolar range [124]. Using a synthetic chemical combinatorial technique, one-bead one-compound (OBOC) method, we identified four heptapeptides (YILIHRN, PTLDPLP, LVLLTRE, and IVFLLVQ) as ligands for the CD21 receptor [125]. The dissociation constants were found to be similar to peptides selected by phage display. Importantly, the peptides retained their biorecognizability towards CD21 receptor after they were conjugated to HPMA copolymers and demonstrated a multivalency effect [125]. Several peptide-targeted HPMA copolymer- drug conjugates displayed anticancer activity [123,126,127]. The combinatorial chemistry approach (OBOC), when combined with a high-stringency screening method, is able to identify peptides with a picomolar affinity [128,129].

3.4.1. Oral, colon-specific delivery of drugs

The development of drug delivery systems capable of selective release of drug in the colon has received much attention. Site-specific delivery to the colon can be achieved by the exploitation of the microbial enzyme activities present predominantly in the colon. The colon has a concentration of microorganisms 5 orders of magnitude greater than the small intestine or stomach. Some of the enzymatic activity produced by microorganisms in the colon, e.g., azoreductase and glycosidase activities do not overlap with the enzymatic activities in the upper GI tract. The azoreductase activities have been studied in detail and used to convert low molecular weight prodrugs into active metabolites in the colon as well as to release active species from water-soluble polymeric carriers [130]. To achieve colon-specific delivery, a (aromatic amino group-containing) drug may be attached to HPMA copolymer side-chains via an aromatic azo bond cleavable by the azoreductase activities present in the colon [51,131-138]. For example, the release of 5-aminosalicylic acid bound to HPMA copolymers via an aromatic azo bond was demonstrated using Streptococcus faecium, an isolated strain of bacteria commonly found in the colon [131], the cecum contents of rats, guinea pigs, and rabbits [133], and in human feces [133].

Recently, we concentrated on the oral delivery of 9-aminocamptothecin (9-AC). First, we attached 9-AC to HPMA copolymers through a spacer containing an aromatic azo bond and amino acid residues [134,135]. It was shown that the aromatic azo bond was cleaved first in vitro [134] and in vivo [135], followed by peptidase-catalyzed cleavage of the amino acid (dipeptide) drug derivative resulting in the release of free 9-AC. However, the cleavage of the peptide drug derivative was not fast enough to achieve high concentrations of free 9-AC in the colon. These results indicated that conjugates containing a spacer with a faster 9-AC release rate need to be designed. To this end, a monomer containing 9-AC, an aromatic azo bond and a 1,6- elimination spacer was designed and synthesized [51]. The combination of the colon-specific aromatic azo bond cleavage and 1,6- elimination reaction resulted in a fast and highly efficient release of unmodified 9-AC from the HPMA copolymer conjugate by cecal contents in vitro, with concomitant stability in simulated upper GI tract conditions. The conjugate possessed a favorable pharmacokinetics [136,137] and was effective in colon cancer models (Fig. 12) [138].

HPMA copolymer-9-aminocamptothecin conjugate. (A) Structure and scheme of release of unmodified 9-AC from HPMA copolymer-9-AC conjugates by a two-step process - rate controlling aromatic azo bond cleavage, followed by fast 1,6-elimination [51]; (B) survival curves of mice bearing human colon carcinoma xenografts treated by 9-AC and P-9-AC at a dose of 3 mg/kg of 9-AC or 9-AC equivalent [138].

3.4.1.1. Targeting in the gastrointestinal tract

Cell-surface glycoproteins reflect the stage of differentiation and maturity of colon epithelial cells. Diseased tissues, carcinomas and pre-cancerous conditions such as inflammatory bowel disease, have altered glycoprotein expression when compared to healthy ones. Consequently, lectins may be used as targeting moieties for polymer-bound drugs [139-141]. Whereas WGA (wheat germ agglutinin) binds to healthy tissues, PNA (peanut agglutinin) binds to diseased tissues. We hypothesized that HPMA copolymer-lectin-drug conjugates could deliver therapeutic agents to diseased tissues by targeting colonic glycoproteins. We examined biorecognition of free and HPMA copolymer-conjugated WGA and PNA and anti-Thomsen-Friedenreich (TF) antigen antibody binding in normal neonatal, adult and diseased rodent tissues, human specimens of inflammation and Barrett's esophagus. Neonatal WGA binding was comparable to the adult, with additional luminal columnar cell binding. PNA binding was more prevalent; luminal columnar cell binding existed during the first 2 1/2 weeks of life. WGA binding was strong in both normal and diseased adult tissues; a slight decrease was noted in disease. PNA binding was minimal in normal tissues; increases were seen in disease. Anti-TF antigen antibody studies showed that PNA was not binding to the antigen. The results suggest that HPMA copolymer-lectin-drug conjugates may provide site-specific treatment of conditions like colitis or Barrett's esophagus [141].

3.5. Subcellular fate and targeting

As discussed above, macromolecular therapeutics are internalized by endocytosis with an ultimate location in the lysosomes. Numerous conjugates were synthesized and evaluated based on this biological rationale. Recently, however, research has been focusing on the identification of different routes of cell entry with the aim to deliver drugs into subcellular compartments different from lysosomes [142- 146]. This direction was mainly driven by attempts to deliver genes or oligonucleotides, i.e., compounds, which may degrade in the lysosomes; however, other rationales may be important: a) the activity of many drugs depends on their subcellular location; and b) the mechanism of action of polymer-bound drugs may be different than that of the free drug. Consequently, manipulation of the subcellular fate of macromolecular therapeutics may result in more effective conjugates.

3.5.1. Mitochondrial targeting

A wide variety of therapeutic agents may benefit by specifically directing them to the mitochondria in tumor cells. To design delivery systems that would enable a combination of tumor and mitochondrial targeting, novel HPMA copolymer-based delivery systems that employ triphenylphosphonium ions as mitochondriotropic agents [147] were developed [142]. Constructs were initially synthesized with fluorescent labels substituting for drug and were used for validation experiments. Microinjection and incubation experiments performed using these fluorescently-labeled constructs confirmed the mitochondrial targeting ability [148]. Subsequently, HPMA copolymer-drug conjugates were synthesized using a photosensitizer mesochlorin e6 (Mce6). Mitochondrial targeting of HPMA copolymer-bound Mce6 enhanced cytotoxicity as compared to non-targeted HPMA copolymer-Mce6 conjugates [142]. Minor modifications may be required to adapt the current design and allow for tumor site-specific mitochondrial targeting of other therapeutic agents.

3.5.2. Hormone-mediated nuclear delivery

Steroid hormone receptors (SHRs) are known to shuttle between the cytoplasm and nucleus of cells. The rationale for using SHRs as vehicles for transporting drugs from the cytoplasm into the nucleus is based upon the binding of the steroid ligand to receptors such as glucocorticoid receptor (GR); the ligand-receptor complex actively migrates to the nucleus [149]. Analysis of the structure of SHRs [150] indicated that hormone structure might be modified without impairing binding to the receptor. Indeed, Rebuffat et al. used steroid receptors as shuttles to facilitate the uptake of transfected DNA into the nucleus of GR-positive cells [151]. Using a similar strategy, we synthesized a hormone-modified photosensitizer (cortisol-modified mesochlorin e6, Cort-Mce6) capable of binding to GR in the cytoplasm and then localizing to the nucleus.

Novel HPMA copolymer-based delivery systems of this derivative were also synthesized [143]. After internalization of a HPMA copolymer-Cort-Mce6 conjugate (via lysosomally degradable GFLG spacer) by endocytosis, Cort-Mce6 was cleaved, translocated to the cytoplasm, bound to the GR, and translocated to the nucleus [143]. To verify that coupling of cortisol to Mce6 maintains the capacity to form a complex with the cytosolic GR resulting in nuclear localization, we investigated the subcellular fate of the modified drug. Cort-Mce6 was monitored in 1471.1 cells transfected with plasmid that expresses green fluorescent protein labeled glucocorticoid receptor (GFP-GR). Cortisol and Mce6 served as positive and negative controls, respectively. GR translocated to the nucleus after attachment of a glucocorticoid analog (e.g., cortisol). The fluorescent GFP label permits the movement of the GR to be monitored in real time. The data (Fig. 13) clearly indicated the time- and concentration-dependent nuclear localization of cortisol-Lys-Mce6 and cortisol. In contrast, cells incubated with Mce6 did not show any alteration in receptor localization following treatment [143].

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Fig. 13

Design of a polymeric construct for targeting to the nucleus. Nuclear targeting ability of cortisol linked to photosensitizer Mce6 was verified using human ovarian SKOV-3 cells transfected with green fluorescence protein (GFP)-tagged glucocorticoid receptor (GR) [143].

3.5.3. Nuclear entry of macromolecules

Once macromolecules are delivered to the cytosol, a number of methods may be employed to traffic them to specific subcellular compartments, such as the nucleus, in order to enhance their therapeutic efficacy [152]. Nuclear localization of macromolecules has been mediated by targeting peptides [145,146].

However, macromolecules (without subcellular targeting moieties) are typically excluded from entering membrane-limited organelles, with the exception of nucleus whose membrane possesses channels that allow the passive uptake of intermediate-sized macromolecules. The NPC (nuclear pore complex) of the nuclear envelope is composed of about 30 different nucleoporin proteins and is the conduit for both nuclear import and export of macromolecules, such as proteins and nucleic acids. In active transport, cargo as large as 40 nm possessing NLS (nuclear localization sequence) or NES (nuclear export sequence) signaling peptides are guided through the channel after binding to nuclear transport receptor proteins [153]. For smaller macromolecules below 10 nm, however, NPCs have been shown to act as non-specific pores that allow exchange between the nucleus and cytoplasm by diffusion [154]. As a conduit for non-biological macromolecules, the NPCs have been shown to transmit PEG-coated gold colloid particles 4-7 nm in diameter [155].

Recently, using fluorescently-labeled HPMA copolymers, we characterized the basic physicochemical properties that determine the distribution and fate of synthetic macromolecules in living cells [152]. Twelve different classes of water-soluble copolymers were created by incorporating eight different functionalized comonomers. These comonomers possessed functional groups with positive or negative charges, or contained short hydrophobic peptides. The copolymers were fractionated to create parallel "ladders" consisting of 10 fractions of narrow polydispersity with molecular weights ranging from 10 to 200 kDa. The intracellular distributions were characterized for copolymer solutions microinjected into the cytoplasm of cultured ovarian carcinoma cells. Even the highest molecular weight HPMA copolymers were shown to quickly and evenly diffuse throughout the cytoplasm and remain excluded from membrane-bound organelles, regardless of composition (Fig. 14). The exceptions were the strongly cationic copolymers, which demonstrated a pronounced localization to microtubules [152]. For all copolymers, nuclear entry was consistent with passive transport through the nuclear pore complex (NPC). Nuclear uptake was shown to be largely dictated by the molecular weight of the copolymers, however, detailed kinetic analyses showed that nuclear import rates were moderately, but significantly, affected by differences in comonomer composition. HPMA copolymers containing amide-terminated phenylalanine -glycine (FG) sequences, analogous to those found in the NPC channel proteins, demonstrated a potential to regulate import to the nuclear compartment. Kinetic analyses showed that 15 kDa copolymers containing GGFG, but not those containing GGLFG, peptide pendant groups altered the size-exclusion characteristics of NPC-mediated nuclear import [152]. One possible explanation is that the GGFG moieties were able to weakly bind to FG-domain crosslinks in a way that altered the dynamics of a putative Nup hydrogel structure, whereas GGLFG peptides would be expected to bind more strongly and not allow a rapid transfer of crosslinks in the hydrogel-like structure of the nucleopore proteins [156]. Example of time-lapse imaging from FRAP (fluorescence recovery after photobleaching). Here, MDAH2774 ovarian cancer cells were microinjected with HPMA copolymer containing 5 mol% of methacrylic acid (Mw=96 kDa) 2 h before experiment. Area was bleached as indicated and cytosolic copolymer re-diffused and restored equilibrium concentration within 3 s [152].

3.6. Mechanism of action and signaling

The hypothesis that free and polymer-bound drugs activate different signaling pathways is based on their internalization mechanisms. Free drug may initiate signaling pathways by interaction with membrane-bound proteins. In contrast, a polymer-bound drug may be hidden in the hydrophilic corona of the random polymer coil and prevented from interaction with membrane proteins during internalization. It may interact with proteins and DNA after being released from the carrier in the lysosomes and translocated into the cytoplasm [87,91]. We shall demonstrate results supporting this hypothesis using two examples: HPMA copolymer conjugates with geldanamycin and DOX.

3.6.1. HPMA copolymer-geldanamycin conjugates

Geldanamycin (GDM), a benzoquinone ansamycin antibiotic, is a heat shock protein inhibitor. It inhibits the capacity of heat shock proteins such as Hsp-90 to form complexes with client oncoproteins. This results in the ubiqitination of cytoplasmic proteins and subsequent proteosomal degradation [157]. However, the development of GDM as a new anticancer drug has been impaired due to its severe toxicity. Structure-activity studies of a large number of GDM derivatives revealed that modification at the 17-position might improve chemical and physicochemical properties, such as stability in serum and water solubility, while maintaining Hsp90-inhibitory activity. Recently, 17-allylamino-17-demethoxy-geldanamycin was evaluated in a phase I clinical study. Significant hepatotoxicity was observed due to lack of tumor cell selectivity [157]. These limitations may be overcome by the use of a drug delivery system.

 

We developed a novel method for the substitution of the 17-methoxy group of GDM to introduce a primary amino group that is useful for conjugation with targeting moieties and HPMA copolymer-based drug carriers [158]. HPMA copolymers containing different AR-GDM (AR=3-aminopropyl (AP), 6-aminohexyl (AH), and 3-amino-2-hydroxypropyl (AP(OH)), attached via a lysosomally degradable GFLG spacer, were synthesized and characterized [159]. The cytotoxic efficacy of HPMA copolymer-AR-GDM conjugates depended on the structure of AR-GDM [160].

 

To verify the hypothesis that P(AP-GDM) [HPMA copolymer-17-(3-aminopropylamino)-17-demethoxy-geldanamycin conjugate] may change the gene expression profiles of low molecular weight GDM derivatives, 32P-macroarray analysis (Clonetech) was employed to evaluate the gene expression profiles in human ovarian carcinoma A2780 cells treated with GDM, AP-GDM and P(AP-GDM) at 2 times 50% cell growth inhibitory concentration (IC50). About 1200 genes related to cancer were evaluated at 6 h and 12 h and three-fold changes in expression were considered significant. Considerable similarities in gene expression profiles were found after AP-GDM and P(AP-GDM) treatments as demonstrated by the hierarchical clustering of the gene expression ratios [91]. However, the outcome was different when individual genes relevant to the mechanism of action of geldanamycin were analyzed. P(AP-GDM)-treated cells showed lower expression of HSP70 and HSP27 compared with AP-GDM up to 12 h. Possibly, internalization pathways and subcellular drug localization of P(AP-GDM), different from low molecular AP-GDM, may modulate the cell stress responses induced by AP-GDM. The results of 32P-macroarray were confirmed by RT-PCR and Western blotting [91]. It is possible that internalization of HPMA copolymer-AP-GDM conjugate via endocytosis may circumvent interactions with external components of the cell, such as plasma membrane, which may be sensitive to stressors and environmental changes (Fig. 15). Similarly, we previously observed that A2780 cells treated with HPMA copolymer-DOX conjugate showed a down-regulation of the HSP70 gene more pronounced than that observed in the cells treated with free DOX [89]. These findings may suggest that conjugation of AP-GDM to HPMA copolymer may be able to modulate the cell stress responses induced by AP-GDM due to differences in its internalization mechanism, subcellular localization, and intracellular concentration gradients [91].

3.7. Cancer: clinical trials

HPMA copolymer-based macromolecular therapeutics have been developed considerably in the last 20 years - numerous conjugates have entered clinical trials for therapeutic validation in the last decade. These include HPMA copolymer-DOX [163-165], HPMA copolymer-DOX-galactosamine [166], HPMA copolymer-camptothecin [167], HPMA copolymer-paclitaxel [168], and HPMA copolymer-platinates [169]. Results from testing of some of these conjugates are promising; hopefully the FDA approval of a first macromolecular therapeutics will occur soon. In Section 4.1 we summarized our ideas on the design principles of second-generation conjugates with enhanced therapeutic potential.

 

3.8. HPMA copolymer conjugates in the treatment of non-cancerous diseases

HPMA copolymer-drug conjugates may be used also for the treatment of diseases other than cancer. We designed bone-targeted HPMA copolymer-conjugated with a well-established bone anabolic agent (prostaglandin E1; PGE1) for the treatment of osteoporosis and other musculoskeletal diseases [50,170-175]. The biorecognition of the conjugates by the skeleton was mediated by an octapeptide of D-aspartic acid (D-Asp8) or alendronate [170,172].

 

This system has the potential to deliver the bone anabolic agent, PGE1, specifically to the hard tissues after systemic administration. Once bound to bone, the PGE1 will be preferentially released at the sites of higher turnover rate (greater osteoclasts activity) via cathepsin K (osteoclast specific) catalyzed hydrolysis of a specific peptide spacer and subsequent 1,6-elimination [50,176]. When given in anabolic dosing range, the released PGE1 will activate corresponding EP receptors on bone cells surface to achieve net bone formation. The main features of the design are HPMA copolymer backbone containing cathepsin K-cleavable oligopeptide side-chains (Gly-Gly-Pro-Nle) terminating in either D-Asp8 or in p-aminoben-zyloxycarbonyl- 1-prostaglandin E1, a PGE1 prodrug (Fig. 17A).

Structure of HPMA copolymer-prostaglandin E1-Asp8 conjugate and mechanism of its cleavage by cathepsin K followed by 1,6-elimination (A) [50]; Bone formation measured in cancellous bone from the lumbar vertebral bodies in ovariectomized rats 4 weeks after a single injection of 10 mg of the conjugate (n=8) (B) [174].

This novel delivery system has several distinct advantages. First of all, it is a double-targeted delivery system, which contains a bone-binding moiety (D-Asp8) and a cathepsin K (osteoclast specific enzyme) specific releasing mechanism. By directing PGE1 specifically to the skeleton, the side effects of systemic administration of the drug would be greatly reduced. Secondly, E-series prostaglandins (PGEs) are powerful anabolic agents in bone, and this delivery system will better target these molecules to sites in the skeleton with a high turnover rate, where new bone formation would be more beneficial. Thirdly, the system permits improved control of drug concentration at the target (bone) site after systemic administration [171,173]. Fourthly, the polymeric carrier can be eliminated from hard tissues and, subsequently, cleared from the body via kidney glomerular filtration. It also offers proper protection of the conjugated PGE1 from metabolism before it reaches bone tissue. Most recently, we observed the preferential deposition of the proposed delivery system to the bone resorption sites in ovariectomized rats; this strongly supports our higher turnover sites/drug-release hypothesis [172]. In vivo experiments on ovariectomized rats have proven the concept. Following a single i.v. administration of the HPMA copolymer-Asp8-PGE1 conjugate to aged, ovariectomized rats, bone formation rates were substantially greater than controls when measured 28 days later (Fig. 17B) [173]. HPMA copolymer conjugates have been also successful in the treatment of rheumatoid arthritis [175,177].

3.9. Methods of synthesis of HPMA copolymer conjugates

The topic of conjugate synthesis will be extensively covered throughout this volume. We have demonstrated several approaches through this chapter. Consequently, we shall just mention some of the important points, which were not covered.

3.9.1. Hydrolytically cleavable bond between drug and HPMA copolymer

Due to the decreased pH in the endosomes and lysosomes, pH-sensitive bonds are suitable for intracellular drug delivery. We have synthesized HPMA copolymer-adriamycin (ADR=DOX) conjugates where ADR was bound via cis-aconityl bond [177] (Fig. 18). The determination of the cytotoxicity of P(aconityl)-ADR toward A2780 sensitive and A2780/AD resistant human ovarian carcinoma cells indicated that the polymer conjugate could overcome the P-glycoprotein efflux pump expressed in A2780/AD cells [178].

3.9.2. Disulfide-linked HPMA copolymer-mesochlorin e6 conjugates

Novel polymeric delivery systems for the photosensitizer mesochlorin e6 (Mce6) were synthesized to overcome problems of systemic toxicity. A disulfide bond was included to allow for quick release ofMce6 from the HPMA copolymer backbone once internalized in tumor tissue (Fig. 19). Synthesized conjugates demonstrated a time-dependent reductive cleavage with an accompanying increase in the quantum yield of singlet oxygen generation on exposure to dithiothreitol. Faster release kinetics and a higher cytotoxicity in SKOV-3 human ovarian carcinoma cells were obtained as compared to polymer conjugate with a proteolytically cleavable glycylphenylalanylleucylglycyl spacer. These novel conjugates hold promise as clinically relevant drug delivery systems for photodynamic therapy of cancer [179].

3.9.3. Attachment of targeting moieties

The chemistry used for attachment of targeting moieties has an impact on the biorecognition of the conjugate. We compared several methods of antibody attachment (see below), investigated how the attachment of antibodies to HPMA copolymers impacts the mechanism of internalization and subcellular trafficking [180] and designed polymerizable antibody fragments [84].

 

3.9.4. Polymerizable antibody fragments

An innovative pathway for the synthesis of targeted polymeric drug delivery systems using polymerizable antibody fragments was designed [84]. A new macromonomer, a polymerizable antibody Fab′ fragment (MA-Fab′) of the OV-TL 16 antibody (IgG1) has been synthesized and copolymerized with HPMA to produce poly(HPMA-co- MA-Fab′) (Fig. 20). The concept of using polymerizable Fab′ fragments as macromonomers provides a new paradigm for the synthesis of targeted polymeric drug delivery systems, and may have unique applications in other areas, such as immunoassays, biosensor technology and affinity chromatography [84].

 

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Fig. 20

Synthesis of polymerizable antibody Fab′ fragment and its copolymerization to produce a targeted HPMA copolymer-Mce6 conjugate [84].

 

3.9.5. Impact of chemistry of attachment of antibodies to HPMA copolymers on the binding affinity of the conjugates

The influence of different methods of coupling the OV-TL16 antibody and its Fab′ fragment to HPMA copolymer-drug (ADR, Mce6) carriers on the binding affinity of the conjugates to the CD47 antigen associated with ovarian carcinoma (OVCAR-3) cells was studied. Three different methods of covalently binding the Ab or Fab′ to polymers were used [181]. Method A: binding via amide bonds formed by aminolysis of active ester groups on the HPMA copolymer-drug (ADR or Mce6) conjugates by amino groups on the antibody; Method B: binding via hydrazone bonds formed by the reaction of aldehyde groups on the oxidized antibody with hydrazo groups on the HPMA copolymer-Mce6 conjugates; Method C: binding via thioether bonds formed by the reaction of sulfhydryl groups of Fab′ fragments with maleimido groups on the side-chain termini of the HPMA copolymer-Mce6 conjugate. Differences in Ka were observed as shown in Fig. 21.

 

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Fig. 21

The influence of different methods of binding the OV-TL16 antibody and its Fab′ fragment to HPMA copolymer-drug (adriamycin {ADR} or meso chlorin e6 mono(N-2-aminoethylamide) {Mce6}] conjugates on the affinity of conjugates to an ovarian carcinoma OVCAR-3 cell associated antigen. Affinity constants Ka (M-1×10-8) are shown for free antibody; antibody bound via amino groups, antibody bound via oxidized saccharide units in the hinge region; and Fab′ fragment bound via thioether bonds [181].

 

3.9.6. Living radical polymerization

The synthesis of HPMA copolymer conjugates, especially their molecular weight distribution, can be controlled by methods of living radical polymerization, RAFT (reversible addition-fragmentation chain transfer) [182] and ATRP (atom transfer radical) polymerizations [183]. For example, HPMA copolymer conjugates containing two drugs and one fluorescent label per macromolecule using RAFT copolymerization were recently synthesized [119; Fig. 10].

 

3.9.7. Attachment of oligonucleotides

Aminolysis of HPMA copolymer precursors can be used for attachment of oligonucleotides to HPMA copolymers. We have attached a 21-mer phosphorothioate oligonucleotide (5′-TTTATAAGGGTCGATGTCCXX-3′) to HPMA copolymers containing GG-ONp and GFLG-ONp (ONp is p-nitrophenoxy) side-chains [184]. The oligonucleotide had a primary amine on the 5′-end and a fluorescein on the 3′-end. The subcellular fate and activity in inhibiting the hepatitis B virus of the HPMA copolymer-phosphorothioate oligonucleotides was studied. Covalently attaching the oligonucleotides to the HPMA copolymers via non-degradable dipeptide GG spacers resulted in sequestering the oligonucleotide in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The degradable HPMA copolymer-oligonucleotide conjugate possessed antiviral activity indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide -HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers [184].

 

3.9.8. Attachment of cell penetrating peptides (CPP)

These peptides, such as the Tat peptide (48GRKKRRQRRR57) which originates from HIV-1 Tat protein, a potent activator of HIV-1 transcription, have been attached using various chemical designs. One approach is to add several amino acid residues, e.g. to produce 48GRKKRRQRRR57YK(FITC)C. The latter may be attached to HPMA copolymer side-chains terminated in maleimide via thioether bonds [185]. We have recently reviewed the biological implications of CPP attachment [144], so we shall not discuss it here.

 

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4. Modification of proteins and vesicular carriers with HPMA (co)polymers

4.1. Modification with semitelechelic poly(HPMA)

Semitelechelic (ST) polymers are linear macromolecules having a reactive functional group at one end of the polymer chain [186]. The single functional group provides the opportunity to conjugate or graft the macromolecule to other species or surfaces. Davis and coworkers have shown that attachment of ST poly(ethylene glycol) (PEG) to therapeutical proteins results in an increase of their resistance to proteolysis, reduction of their antigenicity, and prolongation of intravascular half-life [187]. The extent of protein property changes depends on the degree of PEG substitution and PEG molecular weight [188]. Modification of biomedical surfaces with PEG reduces their biorecognizability and prevents protein adsorption [186].

 

Poly(HPMA) [189] exhibits similar properties as poly(ethylene glycol) [190] when used for modification of enzymes or vesicular carriers. The first report on semitelechelic poly(HPMA) (ST-PHPMA) was published in 1995 [191]. Modification of nanospheres, based on a copolymer of methyl methacrylate, maleic anhydride, and methacrylic acid, with ST-PHPMA resulted in decreased protein adsorption in vitro and increased intravascular half-life, as well as decreased accumulation in the liver, after intravenous administration into rats. The higher the molecular weight of ST-PHPMA, the more pronounced the changes in these properties (Fig. 22). These data seem to indicate the influence of the hydrodynamic thickness of the coating layer on the process of opsonization and capture by Kupffer cells of the liver and macrophages of the spleen [191].

 

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Fig. 22

First synthesis and application of semitelechelic poly(HPMA) to modify surface of nanospheres. (A) Structure; (B) adsorption isotherms of IgG onto surface-modified P (MMA-MA-MAA) nanospheres in 1/15 M saline at 25 °C for 3 h. Each point represents the mean±SD (n=3); (C) body distribution profiles for unmodified and modified [14C]-P(MMA-MA-MAA) nanospheres in rats 24 h after i.v. administration and the correlation with Mn of ST-PHPMA. Each point represents the mean±SD (n=5) [191].

 

Similarly, carboxyl and amino group modification of chymotrypsin with ST-PHPMA-CONHNH2 and ST-PHPMA-COOSu (N-hydroxysuccinimide ester) produced conjugates [189] with comparable properties to PEG-modified chymotrypsin [187]. Ulbrich et al. used ST-PHPMA to modify ribonuclease, chymotrypsin [192], and superoxide dismutase [193]. The proteolytic stability of modified protein increased and their immunogenicity decreased [192,193].

 

4.2. Modification with HPMA copolymers containing multiple reactive side-chains

First protein modified with HPMA copolymers was prepared by the reaction of the copolymer of HPMA with N-methacryloylglycylglycine p-nitrophenyl ester with insulin [28]. The unreacted protein was separated on Sephadex 75; the HPMA copolymer-insulin conjugate exhibited a slower onset and a slight prolongation of hypoglycemic effect in rats when compared to free insulin [28]. Chymotrypsin [29,30] and cobra venom acetylcholinesterase [194] followed.

 

To obtain a better insight into the steric hindrance of the polymer chains on the formation of enzyme-substrate complex, we have studied the hydrolysis of polymeric substrates (HPMA copolymers with oligopeptide side-chains terminated in p-nitroanilide) catalyzed by HPMA copolymer-bound chymotrypsin. The kinetic analysis showed that the hydrolysis of polymer substrates with polymer-bound chymotrypsin led to a decrease in both kcat and kcat/KM, but the relationship between the individual substrates remained intact. Apparently, the steric effects of two independent polymer chains (one bound to substrate, the other to enzyme) were roughly additive [30].

 

Different chemistry was used for the modification of cobra venom acetylcholinesterase [194]. The secondary OH groups of poly(HPMA) (Mw 25-30 kDa) were activated with 4-nitrophenyl chloroformate in dimethylformamide followed by attachment of acetylcholinesterase in borate buffer. The poly(HPMA)-modified acetylcholinesterase demonstrated a 70-fold prolongation of enzyme activity in blood after intravenous injection into mice when compared to unmodified enzyme. The thermoinactivation rate of the polyHPMA-acetylcholinesterase conjugate was 74 times smaller than that of native enzyme (Fig. 23) [194].

 

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Fig. 23

HPMA copolymer-modified acetylcholinesterase. (A) Structure; (B) enhancement of intravascular half-life in mice after i.v. administration of HPMA copolymer-modified enzyme; (C) augmentation of thermal stability of HPMA copolymer-modified enzyme [194].

 

HPMA copolymer with N-methacryloylglycylphenylanylleucylglycine p-nitrophenylester was used for the modification of ribonuclease [195] and superoxide dismutase [193]. No difference in biological activity of conjugates prepared using ST-PHPMA and HPMA copolymers with reactive side-chains was detected [193].

 

There is considerable activity in using HPMA copolymers with reactive side-chains to stabilize complexes of DNA with viruses. This research is covered in the chapter by Seymour in this volume and in our review [196].

 

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5. HPMA in biomaterials design

5.1. HPMA-based hydrogels

First HPMA-based hydrogels were synthesized by crosslinking copolymerization of HPMA and methylene-bis-acrylamide or ethylene- bis-methacrylamide in the early 70s [197]. The kinetic course of copolymerization, interaction parameter polymer-water, the modulus of elasticity, and concentration of elastically effective chains were characterized.

 

Degradable hydrogels containing oligopeptide crosslinks susceptible to chymotrypsin-catalyzed hydrolysis were synthesized by crosslinking HPMA copolymers containing reactive side-chains (terminated in p-nitrophenoxy groups) with oligopeptide (GGY, GFY, GLF, AGVY, and AGFY)-containing diamines [49]. The degradability of hydrogels was dependent on the length and detailed structure of the oligopeptide sequence and on the network density, and thus the equilibrium degree of swelling (Fig. 24). The higher the degree of swelling, the faster the rate of degradation. The degree of swelling also has an impact on surface vs. bulk degradation of the hydrogel. If the enzyme cannot diffuse into the hydrogel interior, only surface degradation takes place. This was demonstrated by a comparison of the degradation of HPMA copolymer crosslinked with AGVY-containing sequences catalyzed by chymotrypsin and HPMA copolymer-bound chymotrypsin. Only surface degradation of the hydrogel was observed in the latter case due to the larger size of polymer-modified enzyme [49]. HPMA-based hydrogels containing the sequence GFYAA in the crosslinks were cleavable with cathepsin B, a lysosomal thiol proteinase [44]. In further experiments, HPMA-based hydrogels with degradable crosslinks were shown to release FITC-dextran and daunomycin during incubation with a mixture of lysosomal enzymes (Tritosomes) or chymotrypsin [198].

Crosslinking copolymerization of HPMA with N,O-dimethacryloyl-hydroxylamine produced hydrolytically degradable hydrogels [199]. These hydrogels were used as a depo for anticancer drug (DOX); results of combination therapy using DOX release from hydrogels followed by antibody-targeted therapy has been effective in the treatment of terminal stages of Bcl1 leukemia in mice [200].

Hennink et al. synthesized ABA triblock copolymers, where block A is thermosensitive poly(HPMA lactate) and block B is PEG. Heating of the copolymer results in the formation of a viscoelastic material, which may be stabilized (crosslinked) by photopolymerization [202]. These materials are suitable for protein delivery [203].

5.2. HPMA-based hybrid hydrogels

Traditional synthetic methods, such as crosslinking copolymerization, crosslinking of polymeric precursors, and polymer-polymer reactions, have produced numerous hydrogel materials with excellent properties [9,204,205]. However, these approaches do not permit a precise control of chain length, sequence, and 3D structure. On the contrary, self-assembly of (macro)molecules into physical hydrogels has the potential to produce precisely defined, hierarchical 3D structures [206-209]. Self-assembly is highly specific and frequently found in biology both in the peptide/protein and DNA worlds.

 

Hybrid hydrogels are usually referred to as hydrogel systems that possess components from at least two distinct classes of molecules, for example, synthetic polymers and biological macromolecules, interconnected either covalently or non-covalently [210]. Conjugation of peptide domains and synthetic polymers may lead to novel materials with properties superior to those of individual components [206-218].

 

5.2.1. Self-assembly of hybrid graft HPMA copolymers into hydrogels mediated by coiled-coil formation

Our research has focused on the factors controlling the self-assembly of graft copolymers composed of a synthetic polymer backbone and a pair of oppositely charged peptide grafts. Two distinct pentaheptad coiled-coil forming peptides (CCE and CCK) were designed to create a dimerization motif and serve as physical crosslinkers [211]. We hypothesized that the antiparallel orientation of heterodimers will contribute to the homogeneity of the self-assembled hybrid hydrogels due to unique interchain dimer formation and decreased steric hindrance of the (synthetic) polymer backbone on the "in-register" alignment of heterodimers. Indeed, equimolar mixtures of the graft copolymers, CCE-P/CCK-P (P is the HPMA copolymer backbone), have been observed to self-assemble into hydrogels in PBS (phosphate buffered saline) solution at neutral pH at concentrations as low as 0.1 wt.% (Fig. 25). Circular dichroism spectroscopy, sedimentation equilibrium experiments, and microrheology data revealed that the self-assembly process corresponded to the two-stranded α-helical coiled-coil formation between CCE and CCK. Moreover, the formation of hybrid hydrogels was reversible. Denaturation of the coiled-coil domains with guanidine hydrochloride (GdnHCl) solutions resulted in disassembly of the hydrogels. Removal of GdnHCl by dialysis caused coiled-coil refolding and hydrogel re-assembly. Similarly, dynamic light scattering data indicated that exposure of the self-assembled hydrogel to a competing peptide (CCK) resulted in a decrease of crosslinking density and partial disassembly of the hydrogel [212]. As expected, concentration of graft copolymers had a significant impact on the kinetics of self-assembly [212] and on the structure and morphology of self-assembled hydrogels [211].

 

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Fig. 25

Self-assembly of HPMA graft copolymers, CCK-P and CCE-P, into hybrid hydrogels mediated by antiparallel heterodimer coiled-coil formation. Two distinct pentaheptad peptides (CCE and CCK) were designed to create a dimerization motif and serve as physical crosslinkers (P is the HPMA copolymer backbone). Aqueous solutions of CCE-P or CCK-P did not form gels. In contrast, gel-like materials were formed from equimolar mixtures of CCE-P/CCK-P at low concentrations [211].

 

5.2.2. Self-assembly of hybrid graft HPMA copolymers into hydrogels mediated by β-sheet formation

The self-assembly of hybrid block- [213] and graft- [214] copolymers composed of polyHPMA and β-sheet motifs was also investigated. β-sheet forming peptides were conjugated to polyHPMA via thiol-maleimide coupling reaction. ST-PHPMA-SH was synthesized via RAFT polymerization of HPMA followed by post-polymerization end-group modification by aminolysis. The diblock copolymer was obtained by the polymer-analogous reaction with N-terminal maleimide-modified β-sheet peptide [213]. The graft copolymer was prepared by attachment of N-terminal cysteine modified β-sheet peptide to poly(HPMA) precursor, which contained pendant maleimide groups [214]. Circular dichroism spectra showed that the strong tendency of the peptide to self-assemble into β-sheets was retained in the copolymers. In addition, β-sheet sensitivity to temperature and pH variations decreased due to poly (HPMA) shielding effect. Atomic force microscopy and small-angle X-ray scattering showed that copolymer had the ability to self-assemble into fibrils. Moreover, transmission electron microscopy showed that poly(HPMA-g-CGGBeta11) fibrils formed matrices with minimal lateral aggregation, dramatically different from the highly aggregated peptide fibrils [214]. This observation coincided with the FTIR results, which suggested poly(HPMA) hindered the twisting of the fibrils formed in copolymer through the antiparallel arrangement of the β-strand peptides. Such materials may be useful in a broad range of biomedical applications, from depots for drug delivery, to scaffolds for cell delivery and tissue engineering.

5.1 Enzyme-Responsive Polymers

Enzymes, which consist of proteins, serve as catalysts for most biochemical reactions, including degradation and oxidization or reduction, under mild conditions, i.e., pH 5-8 and 37 °C (de la Rica, Aili, & Stevens, 2012; Ulijn, 2006; Zelzer et al., 2013). To date, thousands of enzymes have been identified in the human body and their site of action is highly specified (Ulijn, 2006). Thus, the introduction of an enzymatic substrate into polymers affords enzyme-responsive nucleic acid carriers that exert highly localized functions on demand. Among the numerous enzymatic substrates, enzymatically degradable peptide, nucleotide, and ester have been installed into polymer structures (Hu, Zhang, & Liu, 2012). Similar to the disulfide and acid-labile bonds, enzyme-responsive cleavable linkers can elicit site-specific structural change in the polymers and polyplexes for enhanced cellular internalization and the intracellular release of nucleic acids. For example, a substrate peptide for the lysosomal enzyme Cathepsin B was used in the polyplex disintegration and nucleic acid release within the lysosome (Figure 10.11(A)) (Chu, Johnson, & Pun, 2012). In this study, cationic oligomers comprising the Cathepsin B-substrate peptide were copolymerized with N-(2-hydroxypropyl)methacrylamide (HPMA), and the resulting polycation was mixed with pDNA for polyplex preparation. The obtained polyplex significantly facilitated the release of pDNA in the presence of Cathepsin B because of the cleavage of the cationic oligomers from the backbone polymer. Similarly, a substrate peptide for matrix metalloproteinase 2 (MMP-2), which is overexpressed in tumor tissues, was used to design carriers featuring tumor-specific responsibility (Hatakeyama et al., 2011; Yingyuad et al., 2013). The introduction of PEG on the carrier surface via the MMP2-substrate peptide was demonstrated to overcome the aforementioned PEG problem, i.e., the inefficient internalization of nucleic acid carriers by target cells, as well as nontarget cells. Thus, the PEG detachment triggered by tumor-specific enzymes can facilitate the interactions between carriers and cancer cell surface for enhanced tumor accumulation and cancer cell internalization. Notably, when systemically administered into subcutaneous fibrosarcoma tumor-bearing mice, the PEG-detachable siRNA carriers exhibited significantly enhanced tumor accumulation and greater gene silencing in the tumor tissue compared with the PEG-undetachable control without the MMP2-substrate peptide (Hatakeyama et al., 2011).

3.2 Circumventing neutralizing immunity and toxicity

Whereas the innate response to AdV can be a positive feature of Ad vaccines, preexisting neutralizing antibodies against the most common Ad serotypes as well as rapid induction of neutralizing antibodies to AdV and liver toxicity upon systemic administration do represent a hurdle for successful Ad-based immunotherapy. A strategy to reduce Ad neutralization and toxicity is by covalent incorporation of synthetic polypeptide sequences in the viral capsid. One such polypeptide-linking agent is polyethylene glycol (PEG), which in various murine experimental studies has been shown to reduce neutralizing antibody responses (Mok, Palmer, Ng, & Barry, 2005; O'Riordan et al., 1999). Mok et al. (2005) showed that PEGylation could reduce in vitro transduction of target cells but surprisingly had no detrimental effect on in vivo transgene expression or distribution. PEGylation reduced the uptake of AdVs by tissue macrophages and Kupffer cells in vivo, indicating that innate immunity to AdVs was also affected. Similar to PEGylation, coating with the biodegradable polymer polylactic-glycolic acid (PLGA) was shown to reduce AdV neutralization by preexisting antibodies (Beer et al., 1998; Chillón, Lee, Fasbender, & Welsh, 1998). Another polymer coat, based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA), was shown to detarget AdVs and reduce host-vector interactions (Fisher et al., 2001). In addition to the detargeting from CAR-expressing cells and reducing innate immune responses to AdVs, polymer coats like PEG and pHPMA can be linked to targeting moieties like antibodies which allows for specific retargeting of these vectors (Bachtarzi, Stevenson, Šubr, Seymour, & Fisher, 2011; Bachtarzi, Stevenson, Šubr, Ulbrich, et al., 2011; Fisher et al., 2001; Ogawara et al., 2004). In terms of vaccine efficacy, it will be interesting to see whether reduction of recognition by neutralizing antibodies will outweigh the collateral ablation of innate immunogenicity of the modified AdVs.

Photos from the awards and conference are available to view here

Abstract

N-(2-Hydroxypropyl)methacrylamide (HPMA) is a water soluble monomer used in the synthesis of biocompatible and non-immunogenic polymers. In particular, poly(HPMA) can be exploited to sterically stabilize nanoparticles (NPs) suitable for the delivery of lipophilic therapeutics without the concerns related to the use of the polyethylene glycol (PEG), such as allergic reactions and the accelerated blood clearance effect. In addition, the use of the ring opening polymerization (ROP) of a lactone in the presence of an initiator that bears a double bond and a hydroxyl group is a promising way (the so called "macromonomer method") to produce oligoester-based monomers and, in turn, to obtain biodegradable NPs via free radical polymerization. However, HPMA cannot be used as initiator being a secondary alcohol and thus hampering the control over the polymer molecular weight (MW). For this reason, in this work, a novel class of amphiphilic block copolymers that consists of a poly(HPMA) backbone and several short oligo(ε-caprolactone) side chains were produced via the adoption of the reversible addition-fragmentation chain transfer (RAFT) polymerization and the "inversion" of the macromonomer method. The oligoester was first synthesized via the ROP of ε-caprolactone in the presence of a primary alcohol and then attached to HPMA using a succinic acid unit as spacer. The NPs obtained via the self-assembly of these novel block copolymers are designed to degrade into completely water soluble poly(HPMA) chains with a MW lower than the threshold value for the renal excretion. The cytotoxicity of these novel carriers and their ability to load trabectedin, a hydrophobic anticancer therapeutic, were assessed.

Graphical abstract: Poly(HPMA)-based copolymers with biodegradable side chains able to self assemble into nanoparticles

On the other hand, the hydrophilic part of the block copolymer forms the NP shell and provides colloidal stability and protects the loaded drug against protein adsorption and opsonisation. Among the hydrophilic species adopted for the stabilization of these carriers, poly(N-(2-hydroxypropyl)methacrylamide) (poly(HPMA)) is an attractive alternative to the polyethylene glycol (PEG) chains, by far the most adopted for biomedical applications. Besides its biocompatibility and its non-immunogenicity, an advantage of poly(HPMA) over PEG is its secondary alcohol functionality, which allows multiple targeting molecules to be conjugated to the same polymer chain. In addition, poly(HPMA) has been proved to not generate any accelerated blood clearance and allergic reaction compared to the PEG.2-4

The inversion of the macromonomer method is also a good strategy for the functionalization of the final side chain. As an example, when a fixed positive charge is wanted, it is not possible to directly use a quaternary ammonium salt that bears a hydroxyl group (i.e. choline chloride) as initiator in the ROP due to its insolubility in the lactone and in the common organic solvents. For this reason, in the paper of Rooney et al., a tertiary amine bearing an alcohol functionality was adopted as the initiator in the ROP of ε-caprolactone and the double bond was then added to the polyester chain by the reaction with methacryloyl chloride. In the end, the tertiary amine was transformed into the corresponding quaternary ammonium salt via reaction with methyl iodide.10,11 The macromonomer method fails also when HPMA is used as the initiator in the ROP of cyclic esters. This is due to its very low reactivity as a consequence of being a secondary alcohol. In the literature, a direct synthesis of a PLA based macromonomer exploiting the HPMA as the initiator was proposed.12 From a close inspection of the product obtained in that case, it is possible to note a low HPMA conversion that led to a poor control over the macromonomer structure, despite narrowly dispersed polymers could be obtained.

Here we report for the first time the synthesis of a well-defined HPMA-functionalized oligoester via the inversion of the macromonomer method. In particular, the macromonomer is obtained in three steps: (i) the ROP of caprolactone (CL) with benzyl alcohol (BA) as initiator; (ii) the acylation of the -OH bearing PCL chain with succinic anhydride and (iii) the further DCC-mediated esterification with HPMA. In particular, BA has been chosen as the co-catalyst in the ROP since it is a reactive primary alcohol with a high boiling point and because it is less toxic than the alkyl alcohols that are commonly adopted (e.g. dodecanol).13 The resulting macromonomer (hereinafter HPMA-CL5, where 5 is the number of caprolactone units added to the chain) was subsequently used to obtain biodegradable polymeric nanoparticles that are suitable for the drug delivery of poorly water soluble therapeutics. In order to produce these nanoparticles, a two-step sequential RAFT polymerization was adopted. In the first step, a poly(HPMA) water soluble block was obtained to provide steric stabilization to the NPs. Then, the obtained macro RAFT agent was chain extended with the novel lipophilic macromonomer containing HPMA. The obtained amphiphilic diblock copolymer is able to self-assemble in water via a simplified nanoprecipitation method with a rudimental apparatus, thus allowing the NP production shortly before the injection. Furthermore, the copolymer is designed to progressively degrade via the hydrolysis of the ester bonds in the PCL side chains. The mentioned NP degradation behaviour has been studied and resulted into the complete disappearance of the carriers leaving only the water soluble poly(HPMA) backbone, which is expected to be easily excreted by the kidneys due to its low molecular weight.1 The NP cytotoxicity and the ability to load trabectedin, a hydrophobic anticancer compound, have been finally assessed.

2.2 HPMA synthesis

The HPMA was synthesized following a procedure reported in literature.14 Briefly, 25.22 g (238 mmol) of sodium carbonate were suspended in 56 mL of anhydrous dichloromethane (DCM). The temperature was kept at -10 °C using a brine\ice mixture and 16.25 g (216 mmol) of DL-1-amino-2-propanol were added. Finally, 22.60 g (216 mmol, 1 : 1 with respect to DL-1-amino-2-propanol) of freshly distilled methacryloyl chloride were dissolved in 22 mL of DCM and the solution was fed to the reaction flask kept at -10 °C in 45 minutes under vigorous stirring. The mixture was left to equilibrate to room temperature and allowed to react for further 30 minutes. Then, the product was recovered by filtration, dried twice with anhydrous Na2SO4, concentrated under vacuum and left to crystallize at -20 °C overnight. Finally it was washed with cold DCM and recrystallized from acetone. The purity of the product was assessed via proton Nuclear Magnetic Resonance (1H NMR) in deuterium oxide (D2O) using a Bruker 400 MHz spectrometer and the yield was up to 65%.

2.3 HPMA RAFT polymerization

A hydrophilic poly(HPMA) with an average degree of polymerization (DP) equal to 70 was synthesized via RAFT polymerization in a mixture of acetic buffer/ethanol (Scheme 1). Specifically, 1.1 g of HPMA (7.69 mmol), 31 mg of CPA (0.11 mmol, HPMA/CPA mole ratio equal to 70), 10.3 mg of ACVA (0.04 mmol, CPA/ACVA mole ratio equal to 3) were dissolved in 3 g of ethanol and 7 g of acetic buffer and poured in a septa-sealed round bottom flask. The mixture was purged with nitrogen for 30 minutes and heated to 70 °C under stirring. The polymerization was left to proceed for 24 hours, the solvent evaporated under vacuum and the polymer precipitated three times in tetrahydrofuran (THF). Aliquots of the reactive mixture were taken at different times, dried under a nitrogen flow and analyzed via1H NMR (in D2O) and gel permeation chromatography (GPC) to evaluate the monomer conversion and the MW distributions over time. GPC analysis was performed at 35 °C using a mixture of 20/80% acetonitrile (ACN)/0.05 M Na2SO4 as eluent and at a flow rate of 0.5 mL min-1. The instrument (Jasco apparatus) comprises a differential refractive index (RI) detector, three Suprema columns (Polymer Standards Service, particle size 10 μm, pore sizes of 100, 1000, and 3000 Å) and a pre-column. A universal calibration was applied based on polyethylene glycol (PEG) standards.

Scheme 1 Synthesis of hydrophilic poly(HPMA) obtained via RAFT polymerization.

2.4 Synthesis of HPMA-PCL5

As schematically reported in Scheme 2, the lipophilic HPMA-based macromonomer was synthesized by a three-step procedure: (i) ROP of CL with BA as the initiator and tin octoate as the catalyst, (ii) subsequent acylation of the product using succinic anhydride and (iii) DCC-mediated esterification of the obtained oligo(caprolactone) bearing a final carboxyl group with HPMA.

Scheme 2 Synthesis protocol to obtain the HPMA-based lipophilic macromonomer: (a) BA initiated ROP of the ε-caprolactone; (b) acylation of the product using succinic anhydride and (c) DCC-mediated esterification of the carboxylic acid terminated oligo(caprolactone) with the HPMA.

In the first step (Scheme 2a), the ring opening polymerization was conducted with a CL/BA mole ratio and BA/Sn(Oct)2 ratio equal to 5 and 1/200, respectively. 10.55 g of CL (92.4 mmol) and 37 mg of Na2SO4, added to remove water from the system, were heated to 130 °C in a septa-sealed flask under stirring. 2 g (18.5 mmol) of BA were mixed with 37 mg (9.1 × 10-5 mol) of tin octoate and injected in the pre-heated CL containing flask. The polymerization was allowed to proceed for 2.5 hours.

The subsequent acylation (Scheme 2b) was obtained in bulk conditions by adding 1.2 mol equivalents of succinic anhydride to the obtained BA-CL5. Briefly, 2.22 g (22.2 mmol) of succinic anhydride were poured directly into the flask of the previously synthesized oligo(caprolactone) and the mixture was heated to 90 °C and left to react overnight. The final product was purified by dissolution in THF and further precipitation in water.

In the third step (Scheme 2c), HPMA-CL5 was obtained via DCC-mediated esterification between the previously synthesized BA-CL5Q and a 20% molar excess of HPMA, exploiting the DMAP as the catalyst. More in detail, 8.03 g (12 mmol) of BA-CL5Q and 2.09 g (15 mmol) of HPMA were dissolved in 40 mL of anhydrous DCM. The solution was poured in a 100 mL septa-sealed flask kept at 0 °C in a water/ice bath. Finally, 270 mg (2.2 mmol) of DMAP and 2.85 g (14 mmol) of DCC dissolved in 40 mL of anhydrous DCM were fed over a period of 1 h into flask. The reaction was left to equilibrate at room temperature and to react for additional 24 hours. Then the mixture was filtered to remove the white precipitate formed as the co-product of the esterification and the solvent evaporated under vacuum. The dry product was dissolved in THF and precipitated in a large excess of water. The obtained macromonomer was finally recovered as a white waxy solid.

2.5 Synthesis of block copolymers

The amphiphilic block copolymers constituted by a uniform poly(HPMA) backbone were synthesized via the RAFT polymerization in ethanol of the HPMA-CL5 lipophilic macromonomer in the presence of the previously synthesized poly(HPMA), used as the macro-RAFT agent. Four different copolymers were synthesized with a final DP of the hydrophobic block equal to 2, 5, 10 and 20. The tuning of the DP was obtained by simply regulating the initial molar ratio between the HPMA-CL5 macromonomer and the macro-RAFT agent. The nomenclature adopted is composed by the hydrophilic block DP followed by the lipophilic block DP (i.e. 702 is applied to the polymer composed by 70 HPMA units and 2 HPMA-CL5 units). The synthesis procedure is represented in Scheme 3.

Scheme 3 Synthesis of the HPMA-based amphiphilic block copolymers via RAFT polymerization.

In a typical reaction, for the synthesis of 702 (70 units of HPMA and 2 units of HPMA-CL5), 0.647 g of poly(HPMA) and 0.141 g of HPMA-CL5 (HPMA-CL5/poly(HPMA) mole ratio equal to 2) were dissolved in 4 mL of ethanol. The solution was transferred in a septum-sealed flask and purged with nitrogen for 30 minutes. The solution was then heated to 70 °C and 10 mg of ACVA dissolved in 1 mL of ethanol was fed under stirring. The reaction was left to occur for 24 h, after which the same amount of ACVA was added and the reaction went on for additional 24 h. The solvent was then evaporated under vacuum and the polymer purified by precipitation in a large excess of diethyl ether. After drying under a flow of nitrogen, the polymer was collected as a pink powder and characterized via1H NMR in deuterated dimethylsulfoxide (DMSO-d6).

3.1 Synthesis of poly(HPMA)-based macro RAFT agent

The HPMA monomer was synthesized according to the literature.12,14,17 To assess the purity of the produced monomer, 1H NMR analysis in D2O was performed and the obtained spectrum is reported in Fig. S1a.† The spectrum confirms the proper structure of the monomer and its purity after the workup protocol. The synthesized monomer was then polymerized via RAFT polymerization in order to obtain the hydrophilic macromolecular chain transfer agent (macro-CTA) employed in the synthesis of amphiphilic diblock copolymers and as steric stabilizer for the final NPs. The polymerization was carried out in an acetic buffer (pH = 5)/ethanol mixture with a low monomer concentration since it is well known that the propagation kinetic constant (kp) of hydrophilic monomers is higher in water due to its beneficial effect on the transition state of the propagation step and increases as the monomer concentration decreases.18,19 On the other hand, ethanol is necessary to dissolve the CPA. The DP of the polymer can be tuned by controlling the monomer/CPA mole ratio. In this case a DP equal to 70 was targeted. The 1H NMR spectrum of the poly(HPMA) is reported in Fig. S1b.† By evaluating the area of the vinyl signal and that of the signal E, it is possible to assess that a 97% monomer conversion is reached during the polymerization. Further from the NMR spectrum it is possible to evaluate the average DP according to eqn (1).

image file: c7ra11179g-t1.tif (1)

Being A, B and C the signals related to the benzyl chain-end group. From eqn (1) it is possible to demonstrate that the synthesized poly(HPMA) is constituted by an average DP of 75, that is in good accordance with the target (i.e. DP = 70). To demonstrate the living nature of the reaction, the kinetic of the process was evaluated by measuring the monomer conversion (via1H NMR) and the molecular weight of the polymer over time. From Fig. S2a† it is possible to note that a logarithmic trend of the conversion versus time is obtained and a monomer conversion up to 80% is achieved within the first 24 hours of reaction. However, an initial inhibition time of 1.5 h is experienced. As reported in literature, this is a common feature of the RAFT polymerization, especially when the DP is low.20 Analyzing then the molecular weight provided by the GPC, a linear trend is obtained versus the conversion. This confirms the living character of the RAFT polymerization, for which a linear dependency of the molecular weight from the conversion is expected according to eqn (2).

image file: c7ra11179g-t2.tif (2) where χ is the monomer conversion, [M]0 and [CPA]0 are the initial molar concentrations of the monomer and the RAFT agent, Mw M and Mw CPA are the molecular weights of the monomer and the RAFT agent, respectively.

It is possible to observe that CL is very reactive during the ROP, and its conversion reaches values higher than 90% after 4 h for both the HPMA/Sn(Oct)2 mole ratios that were tested. However, the situation is different for the HPMA. In this case, the maximum conversion that can be obtained after 4 h is just of 60%. This very low HPMA conversion can be explained by considering the lower reactivity of a secondary alcohol compared to that of a primary alcohol like 2-hydroxyethyl methacrylate that is commonly used in the ROP of cyclic esters.7,21,23,26-30 This low HPMA conversion inevitably prevents the control of the molecular weight of the produced macromonomer. In fact, a DP (estimated via1H NMR. Fig. S3 in the ESI section†) equal to 9 is obtained instead of the target value of 5. For this reason, in order to obtain a macromonomer with the desired molecular weight and number of CL units, the inversion of the macromonomer synthesis method is required and a three-step process is proposed in this work. The first step is the ROP of the CL exploiting the benzyl alcohol that is a very reactive primary alcohol, as the co-catalyst, in order to produce a well defined oligo (CL) with a target DP equal to 5. The 1H NMR spectrum of this first intermediate is reported in Fig. 2a.

From Fig. 2a it is possible to calculate an average DP value of 4.9, which is close to the target. The second step in the synthesis of the HPMA-CL5 macromonomer is the acylation of the produced BACL5 using succinic anhydride in order to obtain a carboxylic acid-terminated molecule active in the esterification reaction. The acylation was conducted in bulk conditions at 90 °C. At this temperature, the succinic anhydride can be dissolved in the liquid BACL5. The 1H NMR spectrum of the produced BACL5Q is reported in Fig. 2b. From this spectrum, the disappearance of the peak at 3.7 ppm (G in Fig. 2a) confirms the complete functionalization of the BACL5. Further, no residual succinic anhydride is detected after the purification protocol. The synthesized BACL5Q was finally reacted with the HPMA in a DCC-mediated esterification reaction using DMAP as the catalyst. This led to the HPMA-CL5 macromonomer, whose 1H NMR spectrum is reported in Fig. 2c. In this case, the shift of the H peak to 5.15 ppm compared to the original chemical shift of 4 ppm in the HPMA NMR spectrum (peak D in Fig. S1a†) confirms the functionalization of the HPMA and the success of the esterification reaction. The molecular weight distribution of the final macromonomer was also studied via both MALDI-TOF and GPC analysis (Fig. S5 and S6 in the ESI section, respectively†) and an average value of 1179 g mol-1 with a polydispersity equal to 1.14 was obtained, in agreement with the theoretical values.

 

3.3 Synthesis of block copolymers

The poly(HPMA) macro-CTA was chain-extended with the synthesized HPMA-CL5 macromonomer via solution RAFT polymerization conducted in ethanol with a [macro-CTA]/[ACVA] mole ratio equal to 3. Different DPs for the lipophilic block were targeted (i.e. 2, 5, 10 and 20), in order to evaluate the impact of the HPMA-CL5 macromonomer over the self-assembly behaviour of the block copolymers, the NP size and their performance as drug delivery vectors. The monomer conversion during the process was evaluated via1H NMR (Fig. S7 in the ESI section†) and summarized, for the four diblock copolymers, in Table 1.

In conclusion, a poly(HPMA70-b-HPMA-CL5n) biodegradable diblock copolymer constituted by a homogeneous HPMA backbone could be successfully assembled in monodisperse, round shaped NPs using a simplified nanoprecipitation method. This allows producing ready-to-use NPs due to the use of a very little amount of DMSO and rudimental equipment.

The NPs synthesized from the HPMA-based lipophilic macromonomer are composed of short oligo(caprolactone) side chains that can undergo hydrolytic degradation in water. The possibility for a nanovector specifically designed for intravenous administration to degrade leaving no traces is a main concern to avoid polymer accumulation into the bloodstream, as already stressed in the literature.1,8,43 Then, the degradation behaviour of the NPs obtained from the four different diblock copolymers has been studied at 37 °C using PBS (pH = 7.4) as the medium. This is a biologically relevant medium since it is commonly used for intravenous injections. The evolution of the NP size as obtained via DLS over time after the NP incubation at 37 °C is reported in Fig. 4a.

Fig. 4 Evolution of (a) intensity-averaged size, (b) PDI and (c) relative scattering intensity referred to the initial time t = 0 over time for the 702 ( ), 705 ( ), 7010 ( ) and 7020 ( ) NPs in PBS incubated at 37 °C over a month. (d) PSDs for the 702 NPs after the synthesis (black solid line), after 7 days (red dashed line) and after 10 days (blue dotted line) at 37 °C.

From a close inspection, it is possible to observe that the size does not change significantly over 30 days for the copolymers 705, 710 and 7020. This suggests the ability of the poly(HPMA) block to provide colloidal stability to the produced NPs, avoiding their aggregation, over a significant period of time. An exception to this trend is represented by the 702 NPs. In this case their average size abruptly increases after 4 days. It is not surprising that this behaviour is recorded for the copolymer with the lowest molecular weight for its lipophilic block. The degradation of the few oligo(caprolactone) chains leads to an increase in the NP hydrophilicity that, in turn, causes them to significantly swell in water. After having reached a maximum size after 10 days the NPs cannot be detected any longer via DLS. In fact, the complete degradation of the lipophilic monomer leaves a highly hydrophilic poly(HPMA) backbone that is molecularly dissolved in PBS. From Fig. 4b it is clear that the PDI follows a trend that is similar to the NP size, being almost constant for the 705, 7010 and 7020 NPs thus providing a further proof of the colloidal stability provided by the poly(HPMA) block. Again, the PDI significantly increases for the 702 NPs starting from the fourth day, in correspondence with the size increase. An important parameter to be considered when studying NP degradation is the relative scattering intensity, which is directly related to the NP size and concentration.44,45 The trend of this parameter over time is reported in Fig. 4c. For the 702 NPs, the relative scattering intensity rapidly decreases, reaching the 35% of its original value after 10 days. Since the NP size is increasing during this period, this significant reduction in the scattering intensity suggests that the NPs are dissolving and that the degradation is almost complete after 10 days. The degradation is much slower for the 705, 7010 and 7020 NPs, for which the relative scattering intensity reaches the 56%, 65% and 88% of the original value after 30 days, respectively. This progressive reduction in the degradation rate as the lipophilic block DP increases is not surprising. In fact, the higher the lipophilic block DP, the lower the hydration that is expected for the core forming block. This reduces the local water concentration into the core and then slows down the kinetic of the hydrolysis reactions, as already proposed in the literature.23,46-48 Finally, the evolution of the PSD for the 702 NPs is reported in Fig. 4d. Here it is possible to observe that shortly upon the synthesis, a unimodal PSD is obtained. However, after 7 days, a second NP population centered at 260 nm is formed, thus suggesting the NP swelling as a consequence of the degradation and the progressively increasing hydrophilic character of the diblock copolymer. The presence of a second peak can be explained considering that the degradation mechanism is not homogeneous.8 Finally, after 10 days the presence of residual, low size micelles is detected by the DLS before the complete dissolution of the copolymer. This degradation mechanism was already observed and described in the case of NPs obtained via emulsion polymerization from PCL-based macromonomers.8,23,49 However, in this work, the synthesis of the lipophilic part of the block copolymer via a combination of ROP and RAFT polymerization allows to finely tune the NP degradation time in order to obtain drug carriers with an optimized half-life. The progressive release of the oligo(caprolactone) side chains is designed to leave the water soluble, low molecular weight poly(HPMA). The combination of complete solubility and low MW makes the degradation residue easily excreted by the kidneys, which is a key factor in avoiding the risk of polymer accumulation.1

3.5 Cytotoxicity and trabectedin release

To further prove the suitability of the produced NPs as nanovectors aimed at parenteral administration, their in vitro cytotoxicity was evaluated in the case of the TNB cancer MDA-MB-231 cells after exposing them to different concentrations of NP suspension for 48 h. The cell viability expressed as a percentage of the control is reported in Fig. 5a in the case of 702 and 705 NPs.

Fig. 5 (a) Cell viability, expressed as the percentage of viable cells with respect to the control, after 48 h exposure with 702 (grey bars) and 705 (white bars) NPs at different NP concentration in the medium. (b) Evolution of the amount of the released trabectedin from 702 ( ) and 705 ( ) NPs.

It can be noticed that both 702 and 705 proved to be highly biocompatible, even at the highest concentration in the medium. This is not surprising considered that poly(HPMA) is known to be non cytotoxic and that the biocompatibility of polyesters like PLA and PCL has been already demonstrated elsewhere.50-53 However, a slightly decrease in the cell viability (i.e. 88%) is recorded for cells exposed at 0.9 mg mL-1 of 705 NP suspension. This could be due to the sensitivity of the polymer towards hydrolysis of the dithioester of the RAFT agent.43,54

Finally, the ability of the produced amphiphilic NPs to encapsulate and mediate the release of a hydrophobic, anti-tumour drug was studied in the case of trabectedin. The loading efficiency, expressed as the amount of drug entrapped into the NP core compared to that loaded in the process, was equal to 75% and 90% for the 702 and the 705 NPs, respectively (i.e. 5.6 μg and 6.8 μg of trabectedin loaded per mg of polymer). These very high values can be explained considering the simultaneous NP formation and drug loading obtained with the proposed nanoprecipitation method, which leads to a higher loading efficiency compared to a post-synthesis loading process.49,55 Further, the higher lipophilic block MW in the case of the 705 NPs is responsible for a higher loading efficiency compared to the 702 NPs. Once the trabectedin was loaded into the NP core, its release was studied via LC-MS/MS and the results are reported in Fig. 5b. It can be observed that after a 30% initial burst release accomplished within 2 h, the drug was sustainedly released for over 24 h. The trabectedin amount released after this time was the 87% for the 702 NPs and the 75% for the 705 NPs referred to the loaded amount. Again, the higher MW of the lipophilic portion in the case of the 705 NPs accounts for a more efficient drug retention.

Finally, this study proved the ability of the proposed formulation to entrap and sustainedly release a hydrophobic drug over a time scale that is comparable to the NP residence time into the body.56,57 Further, it elucidates the dependence of both drug loading and drug release from the copolymer composition.

4. Conclusion

N-(2-Hydroxypropyl)methacrylamide, known as non toxic, non immunogenic and capable to be functionalized in mild conditions, has been exploited to both synthesize a novel lipophilic and biodegradable HPMA-CL5 macromonomer and to provide steric stability to the NPs produced starting from this macromonomer. In particular, to obtain a well defined and tunable structure for the lipophilic macromonomer, the adoption of an inverse macromonomer method was necessary due to the low reactivity of the HPMA as the co-catalyst in the ROP of cyclic esters. This inverse macromonomer approach comprises three steps: (i) the ROP of the ε-caprolactone in the presence of a reactive primary alcohol (i.e. benzyl alcohol), (ii) the acylation of the product with succinic anhydride and (iii) the DCC-mediated esterification with the HPMA. On the other hand, the direct ROP of the ε-caprolactone, using HPMA as the co-catalyst, proved to lead to a poor HPMA conversion and hence to a poor control over the final macromonomer MW. Then, to produce amphiphilic diblock copolymers able to self-assemble in an aqueous environment forming stable NPs, a poly(HPMA)70 macro-CTA was firstly synthesized via solution RAFT polymerization in ethanol/acetic buffer mixture. This was further chain extended with the HPMA-CL5 macromonomer to obtain a comb-like polymer composed of a homogeneous poly(HPMA) backbone and degradable oligo(caprolactone) pendants. A simplified nanoprecipitation strategy to assemble the final block copolymer in monodisperse core-shell NPs was also introduced. Since it was demonstrated that a high polymer concentration (up to 20% w/w) in the organic phase does not have a detrimental effect on the NP size and PDI, this strategy is promising in the production of ready-to-use nanovectors, avoiding intermediate steps like lyophilization and dialysis. The degradation kinetics of the produced NPs, as well as their ability to encapsulate and mediate the release of a hydrophobic drug was finally studied and related to the copolymer composition.

2. Materials and Methods

2.1. HPMA and HEMA copolymer bead preparation

The synthesis of different batches of HPMA copolymer beads (hydroxyethyl methacrylate/ethyleneglycol methacrylate) has been described elsewhere6,7. Briefly, they were prepared by using a free radical suspension polymerization in an aqueous medium with the use of an organic suspending agent, polyvinyl alcohol (PVA). Dodecanol (DOD) was used as the porogen solvent at varying concentrations. Polymerization was carried out at 70 ºC and stirring at 375 rpm in the presence of the initiator 2,2,azobis-2-methylpropionitrile (AIBN). The HPMA copolymer beads in this study were produced with HPMA:EDMA:DOD in a molar ratio of 1:0.73:1.09. DOD concentration was 41.25% w/w of the total organic weight.

The syntheses of the HEMA copolymer beads (2hydroxyethyl methacrylate/methyl methacrylate/ethyleneglycol methacrylate) have also been described elsewhere6. They were prepared using a free radical suspension polymerization in the presence of an aqueous inorganic magnesium hydroxide gel medium prepared in situ. Dodecanol (DOD) was used as a porogen solvent at several concentrations. The polymerization was carried out at 70 ºC and stirring at 250 rpm in the presence of AIBN as initiator. The HPMA copolymer beads used in this study were produced with HPMA:EDMA:DOD in molar ratio of 1:1.30:0.19:0.40. DOD was used in the concentration of 20% w/w of the total organic percentage.

2.2. Scanning electron microscopy (SEM) preparation

The beads morphology and porosity were evaluated by a 551 A scanning electron microscope (15 KV, Phillips Electronic Instruments, Inc., Mahwah, NJ) coupled with a Polaroid camera model 545 (Polaroid Corp., Cambridge, MA) and 4 × 5 inches film holder. Dry samples were assembled in a double-coated tape mounted on an aluminum stub and gold sputtered for 4.0 min at 10 mA of current and 200 µm Hg of vacuum. Micrographs were exposed for 1/8 s and shot at various magnifications.

2.3. Cell culture preparation

Monkey kidney epithelial fibroblasts (COS-7) were purchased from ATCC (American Type Culture Collection, Rockville, MD). The cells were maintained in Dullbeco's Modified Eagle Medium (DMEM, Gibco BRL Laboratories, Grand Island, NY) and supplemented with 10% fetal bovine serum (FBS), antibiotics (penicillin and streptomycin) and incubated in 5% CO2 incubator at 37 ºC in triplicate. After the fibroblasts reached confluence, the medium was removed and they were washed with sterile phosphate buffer solution (PBS) then, detached using trypsin-EDTA (ethylenediaminotetracetic acid) and suspended in regular medium. To determine their number the cells were exposed to tryptan-blue (Aldrich Chemical Co., Milwaukee, WI) and counted using a hemocytometer. HPMA and HEMA copolymer beads (2.5 µg/mL aqueous suspension, 100 µL) were sterilized in an autoclave, mixed with cells (3 × 105, 100 µL), and seeded into a 6-well polystyrene plate in duplicate and incubated for 72 h. In control experiments, the cells and the beads were separately placed in the medium and incubated. The interactions between the beads and the cells were evaluated under a phase-contrast light microscope (G300 Series Binocular, UNICO, Dayton, NJ).

2.4. Porosity evaluation

Nitrogen sorption analysis (NSA) and mercury intrusion porosimetry (MIP) were used to evaluate the specific surface area (SSA, m2/g), total pore volume (TPV, 103 mL/g), average pore radius (APR, Å) and maximum pore size measured (MPS, Å) of the beads. The gas sorption analyses for specific surface area and pore size determination were performed using the NOVA 1000 analyzer (Quantachrome Corp., Boynton Beach, FL). The vacuum adsorption system consisted of a vacuum pump; a gas supply; a sample container; a calibrated volume; manometer and a coolant. MIP analyses were performed using a mercury contact angle of 140º in the Poremaster 60® (Quantachrome Corp., Boynton Beach, FL). Data was generated by the Quantachrome Poremaster for Windows® software, version 2.03.

3. Results and Discussion

SEM results show that the HPMA copolymer beads (Fig. 1) obtained using the PVA suspending agent were a white fine powder of very small particles (10-20 µm). In contrast, the HEMA copolymer beads (Fig. 2), produced using the Mg(OH)2 suspending agent, appeared as larger white spheres (200 µm). The presence of mesopores (20500 Å diameter) was verified for the HPMA copolymer beads while macropores (> 500 Å diameter) were found in the HEMA copolymer beads.

Both methods of bead preparation rendered hydrophilic copolymers. The HEMA copolymer beads, however, are less hydrophilic than the HPMA copolymer beads because of the presence of the pore size controller MMA, a hydrophobic monomer. Peluso et al.8 have suggested that the hidrophilicity/hydrophobitcityratio of the material surfaces would play an important role in cell growth. The differences in the porous structure of the beads were certainly related to the method of preparation of these particles together with the presence of a cross-linker and a porogen.

NSA and MIP results (Table 1) showed greater specific surface area (SSA, m2/g) and total pore volume (TPV, 10 3mL/g) for the HPMA copolymer beads than the HEMA copolymer beads. An average pore radius (APR) of about ten fold is verified for the HEMA copolymer beads. The results shown in Table 1 are consistent with the SEM results for pore diameter ranges. Both types of beads appear to be porous.

The interactions between the cells and the beads after incubation (n = 3) were visualized using the light microscope and are illustrated in Fig. 3 and Fig. 4 for HPMA and HEMA copolymer beads, respectively. As described earlier, the particle size (~ 20 µm) of HPMA copolymer beads was smaller than the HEMA copolymer beads (~ 200 µm). In comparison to the size of the cells, COS-7 fibroblasts showed a similar particle size relative to HPMA copolymer beads, whereas HEMA copolymer beads were larger than the cells. The density of the cells (3 × 105, 100 µL) was purposely kept low in order to focus on the behavior between the cells and beads.

Figure 3a represents the control for the HPMA copolymer beads. As shown in Fig. 3b, the proliferation of cells revealed that they were able to attach and grow in the presence of the beads and indeed surrounded these particles with a particular agglomerated, confluent pattern. This pattern, nevertheless, was not observed with the large size HEMA copolymer beads (Fig. 4a, control). It is clear from Fig. 4b that the cell-bead association is different from the HPMA copolymer beads. At a higher magnification, as depicted in Fig. 4c, a cell layer coating the bead surface can be visualized. In this case, the cell background density for the HEMA copolymer beads was less when compared to the HPMA copolymer beads (Fig. 3b). This can be explained not only by their larger particle size, but also, by the greater porous texture (larger pore size). The superior adhesive characteristics of the cell onto HEMA copolymer beads may be attributed to the lower degree of hydrophilicity of the polymer beads used, i.e., the additional hydrophobic properties of the chemical structure of the beads, due to the presence of the pore size controller, MMA. Therefore, not only the chemical structure but also the surface roughness affected cell growth, likewise Higuchi et al. have reported5.

Kiremitci, Gürhan, and Piskin1 have observed cell culture in a copolymer using HEMA as a basic monomer, except that it was on a swellable gel based matrix. They showed that cell attachment and growth can be controlled by changing the degree of hydrophilicity, by the addition of MMA, as well as the degree of charge. Jayakrishnan and Chithambara Thanoo9 have reported the manufacture of beads made with the hydrophilic monomer (HEMA) only and cross-linked with EDMA. They reported that the control of porosity is accomplished by the addition of a polymeric diluent in the dispersed phase. In fact, HEMA monomer has been proven to be non-toxic to human cells and affords preparation of beads in a wide size range, which is crucial for many biomedical applications. Nevertheless, the same authors did not report on any cell-bead interactions.

4. Conclusion

The mechanically resistant, cross-linked hydrophilic, copolymers beads of HPMA and HEMA are able to support cell growth. The surface area and pore volume are intimately related to the porosity of the material and greatly influence cell growth. Porosity is an important structural parameter to assess cell growth and attachment. These materials may be used as a cell substrate or for other biomedical applications by selecting proper bead and pore size. Furthermore, toxicity in the form of cell death was not detected in the described conditions, hence, residual solvent or bead synthesis by-products seem to be absent.

Acknowledgments

Financial support for this work granted by Temple University and the fellowship to C.D. Vianna-Soares from Capes/Brasilia/Brazil are gratefully acknowledged. The authors thank Dr. Jennifer Su, Ph.D. for her assistance with the cell experiments. Daniel Gündel,1 Mareli Allmeroth,2 Sarah Reime,1 Rudolf Zentel,2 Oliver Thews1 1Institute of Physiology, Martin Luther University Halle-Wittenberg, Halle (Saale), 2Institute of Organic Chemistry, Johannes Gutenberg-University, Mainz, Germany

Background: Polymeric nanoparticles allow to selectively transport chemotherapeutic drugs to the tumor tissue. These nanocarriers have to be taken up into the cells to release the drug. In addition, tumors often show pathological metabolic characteristics (hypoxia and acidosis) which might affect the polymer endocytosis.

Materials and methods: Six different N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer structures (homopolymer as well as random and block copolymers with lauryl methacrylate containing hydrophobic side chains) varying in molecular weight and size were analyzed in two different tumor models. The cellular uptake of fluorescence-labeled polymers was measured under hypoxic (pO2 ≈1.5 mmHg) and acidic (pH 6.6) conditions. By using specific inhibitors, different endocytotic routes (macropinocytosis and clathrin-mediated, dynamin-dependent, cholesterol-dependent endocytosis) were analyzed separately.

Results: The current results revealed that the polymer uptake depends on the molecular structure, molecular weight and tumor line used. In AT1 cells, the uptake of random copolymer was five times stronger than the homopolymer, whereas in Walker-256 cells, the uptake of all polymers was much stronger, but this was independent of the molecular structure and size. Acidosis increased the uptake of random copolymer in AT1 cells but reduced the intracellular accumulation of homopolymer and block copolymer. Hypoxia reduced the uptake of all polymers in Walker-256 cells. Hydrophilic polymers (homopolymer and block copolymer) were taken up by all endocytotic routes studied, whereas the more lipophilic random copolymer seemed to be taken up preferentially by cholesterol- and dynamin-dependent endocytosis.

Conclusion: The study indicates that numerous parameters of the polymer (structure, size) and of the tumor (perfusion, vascular permeability, pH, pO2) modulate drug delivery, which makes it difficult to select the appropriate polymer for the individual patient.

Keywords: HPMA-LMA copolymers, endocytosis, tumor microenvironment, tumor lines, structure-property relationship

Introduction

Nanoscale drug carriers are a promising approach to transport chemotherapeutic agents specifically to the tumor tissue and to protect normal tissues from toxic side effects. In this study, the enhanced vascular permeability of the tumor vasculature is utilized, which allows large molecular structures (>40 kDa) to leave the bloodstream and to accumulate in the tumor tissue.1 Various chemical structures have been suggested to serve as carriers: liposomes, nanocapsules or polymers.2 However, in liposomes or nanocapsules, the chemotherapeutics are dissolved in the liquid core of the structure; in polymer-drug-conjugated nanoparticles, the drug is directly bound to the polymer backbone. Liposomes or nanocapsules can deliver the drug to the cells by extracellular decomposition of the enveloping structure, by fusion with the cell membrane or by endocytosis. With polymeric nanoparticles, the drug is mostly cleaved enzymatically. Therefore, polymer carriers have to be taken up into the cells by endocytosis.2 Besides phagocytosis, several endocytotic routes have been described for the uptake of free (water soluble) molecules or membrane receptor-bound compounds.2-4 Macropinocytosis is a process for direct drug uptake from the fluid phase during which actin-driven membrane protrusions form a vesicle, which is then incorporated into the cell (macropinosome). During clathrin-mediated endocytosis (CME), intracellular clathrin molecules form a network around a membrane invagination. Afterward, the membrane scission protein dynamin (a guanosine triphosphate hydrolase [GTPase]) unhitches the clathrin-coated pit from the cell membrane. A clathrin-independent mechanism is the caveolae-mediated endocytosis, which is cholesterol-dependent requiring cholesterol-rich microdomains (rafts) in the cell membrane associated with caveolin-1 to form caveolar vesicles. In this process, the detachment of the vesicle from the cell membrane probably also requires dynamin.2,3,5 But cholesterol is also essential for CME.6

 

Suitable chemical structures for the design of polymer nanoparticles should be nontoxic, nonimmunogenic and degradable. One promising compound fulfilling these requirements is poly-N-(2-hydroxypropyl)methacrylamide (p[HPMA]) polymers that have been already tested in preclinical as well as clinical studies.7-9 Further development of these hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) homopolymers led to the design of copolymers containing lipophilic lauryl methacrylate (LMA) segments within the HPMA backbone. These HPMA-LMA copolymers (p(HPMA)-co-p(LMA) and p(HPMA)-b-p(LMA) copolymers) have been shown to improve the drug transport through the blood-brain barrier.10,11 The lipophilic segments orientate themselves in the core, whereas the hydrophilic HPMA segments are located in the shell. Depending on the chemical structure of these molecules and on the ratio of lipophilic LMA segments in the HPMA backbone, the HPMA-LMA copolymers form self-assembled structures in which several polymer chains are aggregated (micellar superstructures). In random p(HPMA)-co-p(LMA) copolymers, most of the LMA elements are oriented in the core; however, numerous lipophilic segments are also located on the surface of the polymer nanoparticle.10-13 The p(HPMA)-b-p(LMA) block copolymers, which are synthesized from HPMA and HPMA-LMA oligomers (blocks), show a strict lipophilic core and a hydrophilic surface.11,12,14,15 It was found that homopolymers and random and block copolymers show profoundly different biological behavior in vivo.13-16 On the macroscopic scale, homopolymer and block copolymers accumulated preferentially in the spleen and liver, whereas the random copolymer stayed more pronounced in the blood. When analyzing the overall accumulation in solid-growing AT1 prostate and Walker-256 mammary carcinomas, the intratumoral concentration of the random copolymer was more than twice the level of the homopolymer or the block copolymer. However, in these studies,14,15 only the whole tissue concentration was measured, and it could not be differentiated whether the polymers entered the cells or stayed in the extracellular space. For this reason, the cellular uptake in these two cell lines was analyzed in the current study.

 

Finally, it is well known that the metabolic microenvironment of tumors is profoundly different from that in normal tissues. Many tumors show hypoxic/anoxic regions, and the glycolytic metabolism is intensified leading to a marked extracellular acidosis with pH values down to 6.17,18 It has been shown that hypoxia can decelerate different routes of endocytosis (CME and clathrin-independent endocytosis) mostly via the key regulator hypoxia-inducible factor (HIF).19-21 An acidic extracellular environment has also been described to modulate endocytosis/phagocytosis in macrophages.22-24 On the other hand, studies revealed that the chemical structure of polymers may also play a role for their endocytotic uptake. Liu et al25 demonstrated that HPMA-based polymers containing alkaline side chains, eg, 2-(N, N-dimethylamino) ethyl methacrylate (positive ζ potential), were taken up by cells much stronger than polymers containing acidic adducts, eg, methacrylic acid (negative ζ potential). This mechanism could be of importance in particular in an acidic extracellular environment. But it remains unclear whether the extracellular pH is also important for the uptake of polymers containing lipophilic segments such as in the HPMA-LMA copolymers studied.

 

Hypoxia (as a common phenomenon in solid tumors) can limit the ATP yield in the tumor cell, which is the essential energy source for active uptake processes. On the other hand, extracellular acidosis could affect the interaction of the polymer with the cell membrane (eg, by changing the surface charge of the molecule). For these reasons, the aim of the current study was to analyze the impact of hypoxia and extracellular acidosis (which are common characteristics of solid-growing tumors) on the uptake of HPMA-based polymer nanoparticles in tumor cells. Therefore, fluorescent HPMA homopolymers as well as random p(HPMA)-co-p(LMA) and p(HPMA)-b-p(LMA) block copolymers with low and high molecular weight were synthetized, and the polymer uptake was measured in two different cancer cell lines (AT1 and Walker-256 cells), which have already been studied as solid tumors in vivo.15,16 With these models, it was studied whether extracellular acidosis with pH 6.6 or hypoxic conditions (pO2 ≈1.5 mmHg) affects the uptake of the different HPMA-based polymers. Finally, to analyze the different endocytotic routes of polymer uptake, inhibitors of specific mechanisms were used to distinguish between macropinocytosis and clathrin-mediated, dynamin-dependent and cholesterol-dependent endocytosis.

Materials and methods

Materials

All chemicals were of analytical grade and obtained from Sigma-Aldrich Co. (St Louis, MO, USA) and Acros Organics (Thermo Fisher Scientific, Waltham, MA, USA).

Polymer synthesis

The synthesis of HPMA homopolymers as well as p(HPMA)-co-p(LMA) and p(HPMA)-b-p(LMA) copolymers (Scheme 1) has been described previously together with the characteristics of the polymers, which is summarized in Table 1.14,15 Hydrodynamic radii were determined by fluorescence correlation spectroscopy using a commercial setup (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). To ensure the comparability with the biological situation (cell culture and in vivo), polymers were dissolved in isotonic saline for FCS measurements.

Scheme 1 Chemical structure of (A) the p(HPMA) homopolymers and (B) the p(HPMA)-co-p(LMA) copolymers used.

Abbreviations: HPMA, N-(2-hydroxypropyl)methacrylamide; LMA, lauryl methacrylate.

Table 1 Analytical data of HPMA homopolymers as well as of random p(HPMA)-co-p(LMA) copolymer and p(HPMA)-co-p(LMA) block copolymers with low and high molecular weight, respectively14,15

Notes: aDispersity (polydispersity index) determined by GPC in THF as solvent. bHydrodynamic radii were determined in isotonic saline by fluorescence correlation spectroscopy. cMonomer ratio determined by 1H NMR spectroscopy after a polymer analogous reaction with 2-hydroxypropylamine. dCalculated from the molecular weights of the reactive ester polymer precursors as determined by GPC in THF as solvent.

Abbreviations: GPC, gel permeation chromatography; HPMA, N-(2-hydroxypropyl)methacrylamide; LMA, lauryl methacrylate; NMR, nuclear magnetic resonance; THF, tetrahydrofuran.

The polymers were labeled with Oregon Green-480 (excitation 485 nm, emission 532 nm). This fluorochrome has the advantage that its fluorescent is not pH dependent, which is essential for experiments, in which the extracellular pH was varied.

Tumor models

All studies were performed with two tumor cell lines of the rat: 1) Walker-256 mammary carcinoma (#CCL-38, American Type Culture Collection [ATCC], Manassas, VA, USA) and 2) subline AT1 of the Dunning prostate carcinoma R3327 (#500121, CLS Cell Lines Service GmbH, Eppelheim, Germany). Both cell lines were grown in culture in RPMI medium supplemented with 10% fetal calf serum (FCS) and with 10 mM L-glutamine for Walker-256 cells at 37°C under a humidified 5% CO2 atmosphere and sub-cultivated twice per week.

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