Mucoadhesive drug carrier based on interpolymer complex of poly(vinyl pyrrolidone) and poly(acrylic acid) prepared by template polymerization.

To develop a new mucoadhesive drug carrier, poly(vinyl pyrrolidone) (PVP)/poly(acrylic acid) (PAA) interpolymer complexes were prepared by the template polymerization of acrylic acid using PVP as a template polymer. Fourier transform infrared results showed that the interpolymer complexes were formed by hydrogen bonds between the carboxyl groups of PAA and the carbonyl groups of PVP. The adhesive forces of the PVP/PAA interpolymer complexes were higher than that of commercial Carbopol 971. Moreover, the adhesive force and the release rate can be controlled by changing the mole ratios of PVP and PAA. The release rates of ketoprofen from the PVP/PAA interpolymer complexes showed pH-dependency, and were slower at lower pH. The release rate of ketoprofen from the complex seemed to be mainly controlled by the dissolution rate of the complex above a pK(a) of PAA (4.75) and by the diffusion rate below the pK(a). The prepared complex appears to be an adequate carrier for the mucoadhesive drug delivery system.     

Leakage of enteric (Eudragit L)-coated dosage forms in simulated gastric juice in the presence of poly(ethylene glycol).

Poly(methacrylic acid) (PMAA) and related copolymers strongly interact with poly(ethylene glycol) (PEG) in acidic fluids. Due to the in vitro experiments presented in this paper, there is a clear indication for a drug-drug interaction in vivo between PEG solutions, e.g., commercially available laxatives, and dosage forms with PMAA-based enteric-coatings (Eudragit L). In these studies, enteric-coated tablets did not fulfil the pharmacopoeias' criteria of the disintegration test if PEGs were present in the simulated gastric juice. Drug substances which are known to be unstable in acidic media or which can cause gastric irritation were released from their enteric-coated dosage forms in acidic PEG media (pH 1). Various drug dosage forms, single and multiple unit systems, were tested. They show higher and faster drug release in the presence of PEG. To get insight into the mechanism of the interaction, experiments and theoretical calculations were performed which reveal that PEGs with high molecular weight show stronger interactions with PMAA coatings indicating a contribution of hydrophobic interactions to the occurring intermolecular forces. Hydrogen bonds can be build between each monomeric unit of PEG and the acidic sequences of the copolymer.     

Drug release from poly(acrylic acid) grafted poly(vinylidene fluoride) membrane bags in the gastrointestinal tract in the rat and dog.

Stomach-specific drug delivery systems would be of value in treating diseases of the upper gastrointestinal tract. The present study measured in vitro and in vivo drug release from pH-sensitive membrane bags, constructed of poly(acrylic acid) grafted onto a poly(vinylidene fluoride) (PAA-PVDF) membrane, which might be suitable for stomach-specific drug delivery. The used model drugs were propranolol-HCl (1.0 mg) and FITC-dextran MW 4400 (1.0 mg). Drug release in vivo was studied by inserting membrane bags into the stomach and proximal duodenum of anesthetized rats and dogs. At 30 and 180 min, the bags were removed from the lumens and residual drug content was determined. The release of either propranolol or FITC-dextran were comparable in both stomach and duodenum, showing that in vivo drug release did not depend on environmental pH. In vitro results suggested that these results could be explained by interactions between PAA and the mucous layers of the stomach and duodenum.     

Release of PEGylated granulocyte-macrophage colony-stimulating factor from chitosan/glycerol films.

We have prepared a new formulation for mucosal delivery of GM-CSF or PEGylated GM-CSF based on a chitosan carrier plus added glycerol to control the rate of release of the protein. Thin dry films comprised of various weight ratios of chitosan to glycerol and containing either granulocyte-macrophage colony-stimulating factor (GM-CSF) or PEGylated GM-CSF, PEG-(GM-CSF), were prepared. The amount of GM-CSF or PEG-(GM-CSF) released from the chitosan/glycerol films was determined using size exclusion high performance liquid chromatography (HPLC-SEC). The amount of PEG-(GM-CSF) released from the films decreased with an increase in the amount of glycerol present in the film. In parallel with this, films with higher glycerol content exhibited a lower degree of equilibrium swelling when immersed in release media. pH measurements of the release media and analysis of the dried films by Fourier-transform infrared spectroscopy (FTIR) suggested that the amount of residual acetic acid in the dry films decreased as the glycerol content increased. This indicates that glycerol may act by displacing and releasing bound acetic acid from the chitosan molecules, resulting in chitosan--glycerol hydrogen bond formation as the film dries. Further, it was found that the release rate and the amount of PEG-(GM-CSF) released decreased with increasing molecular weight of the conjugated PEG. This effect was not observed with films containing physical mixtures of PEG and GM-CSF. The decrease in the fraction of PEG-(GM-CSF) released with increasing PEG molecular weight is believed to be due to the increased steric hindrance of the PEGylated protein molecule during its diffusion out of the swollen chitosan/glycerol film.     

PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery.

We investigated the in vitro and in vivo properties of DNA/transferrin-polyethylenimine (800 kDa) complexes before and after covalent coupling of poly(ethylene glycol) (PEG). Upon incubation with plasma, the positively charged non-PEGylated DNA complexes form aggregates. Plasma proteins such as IgM, fibrinogen, fibronectin and complement C3 were found to bind to non-PEGylated DNA complexes. At DNA concentrations relevant for in vivo gene delivery a strong aggregation of erythrocytes was also observed. PEGylation of the complexes strongly reduces plasma protein binding and erythrocyte aggregation. Furthermore, PEGylated complex size was stabilized and had a reduced surface charge. Prolonged circulation in the blood of the PEGylated complexes was also observed when injected intravenously. In tumor bearing mice, application of non-PEGylated complexes through the tail vein resulted in reporter gene expression in tail and lung, but severe toxicity was observed in some mice. In contrast, PEGylated complexes mediated reporter gene transfer to the tumor without significant toxicity.     

A poly(ethylene glycol)-based surfactant for formulation of drug-loaded mucus penetrating particles

Mucosal surfaces are protected by a highly viscoelastic and adhesive mucus layer that traps most foreign particles, including conventional drug and gene carriers. Trapped particles are eliminated on the order of seconds to hours by mucus clearance mechanisms, precluding sustained and targeted drug and nucleic acid delivery to mucosal tissues. We have previously shown that polymeric coatings that minimize adhesive interactions with mucus constituents lead to particles that rapidly penetrate human mucus secretions. Nevertheless, a particular challenge in formulating drug-loaded mucus penetrating particles (MPP) is that many commonly used surfactants are either mucoadhesive, or do not facilitate efficient drug encapsulation. We tested a novel surfactant molecule for particle formulation composed of Vitamin E conjugated to 5 kDa poly(ethylene glycol) (VP5k). We show that VP5k-coated poly(lactide-co-glycolide) (PLGA) nanoparticles rapidly penetrate human cervicovaginal mucus, whereas PLGA nanoparticles coated with polyvinyl alcohol or Vitamin E conjugated to 1 kDa PEG were trapped. Importantly, VP5k facilitated high loading of paclitaxel, a frontline chemo drug, into PLGA MPP, with controlled release for at least 4 days and negligible burst release. Our results offer a promising new method for engineering biodegradable, drug-loaded MPP for sustained and targeted delivery of therapeutics at mucosal surfaces.     

PEGylated bioactive molecules in biodegradable polymer microparticles

Injectable peptide and oligonucleotide biotherapeutics offer great promise for treatment of serious chronic diseases but almost always need further formulation work to increase stability and circulation lifetimes. Covalent attachment of poly(ethylene glycol) (PEG) will increase circulation lifetimes up to a week or so and decrease degradation in favorable cases. Encapsulation in biodegradable polymer microparticles has been highly successful, mostly for peptides to provide sustained release up to several months after injection. Although products are on the market using these technologies, PEGylation and microparticle encapsulation each have drawbacks that prevent more widespread use. When they are combined, the limitations of one technology may be resolved by the other. Work in several laboratories on encapsulation of PEGylated bioactive molecules has revealed a synergy. Activity reduction and restricted circulation lifetimes for PEGylated bioactive agents is addressed by microencapsulation and using a lower PEG molecular weight. Chemical degradation, excessive burst release and limited drug content are typical problems for microparticles that are ameliorated by using PEGylated actives. The case for synergy between PEGylation and microencapsulation is illustrated in this review by work with several proteins and peptides including insulin, and the oligonucleotide therapeutic, pegaptanib.     

PEGylated bioactive molecules in biodegradable polymer microparticles

Injectable peptide and oligonucleotide biotherapeutics offer great promise for treatment of serious chronic diseases but almost always need further formulation work to increase stability and circulation lifetimes. Covalent attachment of poly(ethylene glycol) (PEG) will increase circulation lifetimes up to a week or so and decrease degradation in favorable cases. Encapsulation in biodegradable polymer microparticles has been highly successful, mostly for peptides to provide sustained release up to several months after injection. Although products are on the market using these technologies, PEGylation and microparticle encapsulation each have drawbacks that prevent more widespread use. When they are combined, the limitations of one technology may be resolved by the other. Work in several laboratories on encapsulation of PEGylated bioactive molecules has revealed a synergy. Activity reduction and restricted circulation lifetimes for PEGylated bioactive agents is addressed by microencapsulation and using a lower PEG molecular weight. Chemical degradation, excessive burst release and limited drug content are typical problems for microparticles that are ameliorated by using PEGylated actives. The case for synergy between PEGylation and microencapsulation is illustrated in this review by work with several proteins and peptides including insulin, and the oligonucleotide therapeutic, pegaptanib.     

Preparation, characterization and properties of sterically stabilized paclitaxel-containing liposomes.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, approved by the FDA for the treatment of ovarian and breast cancers. Due to its low solubility in water, it is clinically administered dissolved in Cremophor EL, (polyethoxylated castor oil) and ethanol, which cause serious side effects. Inclusion of paclitaxel in liposomal formulations has proved to be a good approach to eliminating this vehicle and improving the drug's antitumor efficacy. We prepared different conventional and PEGylated liposomes containing paclitaxel and determined encapsulation efficiency, physical stability and drug leakage in human plasma. The best conventional liposome formulation was composed of ePC/PG 9:1, while for PEGylated liposomes the best composition was ePC/PG/CHOL/PEG(5000)-DPPE 9:1:2:0.7. PEGylated liposomes were found to be less stable during storage than the corresponding conventional liposomes and to have lower drug release in human plasma at 37 degrees C. In vitro cytotoxic activities were evaluated on HT-29 human colon adenocarcinoma and MeWo melanoma cell lines. After 2 and 48 h, conventional liposomes had the same cytotoxicity as free paclitaxel, while PEGylated liposomes were as active as free drug, only after 48 h. Pharmacokinetics and biodistribution were evaluated in Balb/c mice after i.v. injection of paclitaxel, formulated in Cremophor EL or in conventional or in PEGylated liposomes. Encapsulation of paclitaxel in conventional liposomes produced marked differences over the free drug pharmacokinetics. PEGylated liposomes were long-circulating liposomes, with an increased t(1/2) beta 48.6 h, against t(1/2) beta 9.27 h of conventional liposomes. Biodistribution studies showed a considerable decrease in drug uptake in MPS-containing organs (liver and spleen) at 0.5 and 3 h after injection with PEGylated compared to conventional liposomes.     

Novel powder formulations for controlled delivery of poorly soluble anticancer drug: application and investigation of TPGS and PEG in spray-dried particulate system.

Biodegradable poly (lactic-co-glycolic acid) (PLGA), D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) and/or polyethylene glycol (PEG) were combined as pharmaceutical excipient to fabricate microparticles containing sparingly soluble drug paclitaxel by spray-drying technique with successful achievement. The effect of formulation variety on particle morphology, surface composition, thermal property, drug entrapped capability, and drug release profile was investigated. The result indicated that the use of the appropriate mixtures of PLGA, TPGS and/or PEG produced paclitaxel-loaded microparticles characterised by acceptable pharmaceutical properties. Atomic force microcopy (AFM) and scanning electron microscopy (SEM) showed that the produced microparticles were spherical in shape with dimples or pores. The particle size ranged from 0.88 to 2.44 microm with narrow distribution. The combination of TPGS and PEG in the formulation resulted in a narrow particle size distribution in general although the influence of the formulation on the particle size was not significant. Differential scanning calorimetry (DSC) study implied that all those components in consideration were compatible well in the blend formulation systems. The paclitaxel entrapped in the particles existed in an amorphous or disordered-crystalline status in the matrices and was independent of the PLGA/TPGS/PEG ratio. X-ray photoelectron spectroscope (XPS) analysis revealed that after incorporation the particle's surface was dominated with PLGA due to its hydrophobic property. The formulation variety had an important impact on the drug release that was reduced with the presence of large fraction of TPGS resulting from a strong hydrophobic interaction between various matrix materials and the drug inside the particle. A zero order release could be yielded by optimising the ratio of PLGA/TPGS/PEG. The combination of PLGA/TPGS/PEG as safe pharmaceutical excipient to formulate particulate delivery system is beneficial in improving the pharmaceutical properties for further powder dosage application.     

Biodegradable poly(ethylenimine) for plasmid DNA delivery

Poly(ethylenimine) (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on the molecular weight. Synthesis of cationic copolymers derived from the low molecular weight of PEI and hydrophilic poly(ethylene glycol) (PEG), which are water soluble and degradable under physiological conditions, was investigated for plasmid delivery. Hydrophilic PEG is expected to reduce the toxicity of the copolymer, improve the poor solubility of the PEI and DNA complexes, and help to introduce degradable bonds by reaction with the primary amines in the PEI. Considering the dependence of transfection efficiency and cytotoxicity on the molecular weight of the PEI, high transfection efficiency is expected from an increased molecular weight of the copolymer and low cytotoxicity from the introduction of PEG and the degradation of the copolymer into low molecular weight PEIs. Reaction conditions were carefully controlled to produce water soluble copolymers. Results from a gel retardation assay and zetapotentiometer indicated that complete neutralization of the complexes was achieved at the charge ratios of copolymer/pSV-β-gal plasmid from 0.8 to 1.0 with the mean particle size of the polyplexes ranging from 129.8±0.9 to 151.8±3.4 nm. In vitro transfection efficiency of the synthesized copolymer increased up to three times higher than that of starting low molecular weight PEI, while the cell viability was maintained over 80%.     

An oral delivery device based on self-folding hydrogels.

A self-folding miniature device has been developed to provide enhanced mucoadhesion, drug protection, and targeted unidirectional delivery. The main part of the device is a finger like bilayered structure composed of two bonded layers. One is a pH-sensitive hydrogel based on crosslinked poly(methyacrylic acid) (PMAA) that swells significantly when in contact with body fluids, while the other is a non-swelling layer based on poly(hydroxyethyl methacrylate) (PHEMA). A mucoadhesive drug layer is attached on the bilayer. Thus, the self-folding device first attaches to the mucus and then curls into the mucus due to the different swelling of the bilayered structure, leading to enhanced mucoadhesion. The non-swelling PHEMA layer can also serve as a diffusion barrier, minimizing any drug leakage in the intestine. The resulting unidirectional release provides improved drug transport through the mucosal epithelium. The functionality of this device is successfully demonstrated in vitro using a porcine small intestine.     

Release of 5-amino salicylic acid from acrylic type polymeric prodrugs designed for colon-specific drug delivery.

New acrylic type polymeric systems having degradable ester or amide bonds linked to the bioactive agent 5-amino salicylic acid (5-ASA), were prepared and evaluated as materials for colon-specific drug delivery. Methacryloyloxyethyl 5-amino salicylate (MOES), and N-methacryloylaminoethyl 5-amino salicylamide (MAES) were prepared as the polymerizable derivatives of 5-ASA using activated ester methodology. The drug-containing monomers were free radically copolymerized with methacrylic acid or hydroxyethyl methacrylate, utilizing azobisisobutyronitrile as initiator. The polymer bearing 5-ASA units as side substituents of the acrylic backbone were obtained in the form of poly pendent esters or poly pendent amides. The drug release studies were performed by hydrolysis in buffered solutions (pH 1, 7.2, 8.5), or simulated intestinal fluid containing pancreatin to measure the chemical degradation expected to occur in the intestinal tract. The release profiles indicated that the hydrolytic behavior of polymers strongly depends on their degree of swelling, type of comonomer, and the nature of hydrolyzable bond. Implication of the results for use of these polymers for colon targeting are discussed.     

Preparation and in vitro characterization of poly(acrylic acid)-cysteine microparticles.

The purpose of the present study was to prepare and characterize a novel mucoadhesive microparticulate drug delivery system. Microparticles were prepared by the solvent evaporation emulsion technique using a poly(acrylic acid)-cysteine conjugate of an average molecular mass of 450 kDa with an amount of 308 micromol thiol groups per gram polymer. The cross-linking of thiol groups via the formation of disulfide bonds during this preparation process was pH-controlled. The resulting microparticles were characterized with regard to the degree of cross-linking and the amount of remaining free thiol groups, shape, size distribution and stability. Furthermore, the drug release behaviour using bromelain as model drug and the mucoadhesive properties were evaluated.Results demonstrated that the higher the pH of the aqueous phase was during the preparation process, the higher was the degree of cross-linking within the particles. However, even at pH 9, 8.9+/-2.2% of free thiol groups remained on the microparticles. Particles were of spherical and partially porous structure and had a main size in the range of 20-60 microm with a center at 35 microm. Because of the formation of disulfide bonds within the particles, they did not disintegrate under physiological conditions within 48 h. In addition, a controlled drug release of bromelain was achieved. Due to the immobilization of thiol groups on poly(acrylic acid), the mucoadhesive properties of the corresponding microparticles were improved threefold.These features should render poly(acrylic acid)-cysteine conjugate microparticles useful as drug delivery system providing a prolonged residence time on mucosal epithelia.     

Engineering of NIR fluorescent PEGylated poly(RGD) proteinoid polymers and nanoparticles for drug delivery applications in chicken embryo and mouse models

Proteinoids are non-toxic biodegradable polymers based on thermal step-growth polymerization of natural or synthetic amino acids. Hollow proteinoid nanoparticles (NPs) may then be formed via a self-assembly process of the proteinoid polymers in an aqueous solution. In the present article polymers and NPs based on d-arginine, glycine and l-aspartic acid, poly(R^DGD), were synthesized for tumor targeting, particularly due to the high affinity of the RGD motif to areas of angiogenesis. Near IR fluorescent P(R^DGD) NPs were prepared by encapsulating the fluorescent NIR dye indocyanine green (ICG) within the formed P(R^DGD) NPs. Here, we investigate the effect of the covalent conjugation of polyethylene glycol (PEG), with different molecular weights, to the surface of the near IR encapsulated P(R^DGD) NPs on the release of the dye to human serum due to bio-degradation of the proteinoid NPs and on the uptake by tumors. This work illustrates that the release of the encapsulated ICG from the non-PEGylated NPs is significantly faster than for that observed for the PEGylated NPs, and that the higher molecular weight is the bound PEG spacer the slower is the dye release profile. In addition, in a chicken embryo model, the non-PEGylated ICG-encapsulated P(R^DGD) NPs exhibited a higher uptake in the tumor region in comparison to the PEGylated ICG-encapsulated P(R^DGD) NPs. However, in a tumor xenograft mouse model, which enables a prolonged experiment, the importance of the PEG is clearly noticeable, when a high concentration of PEGylated P(R^DGD) NPs was accumulated in the area of the tumor compared to the non-PEGylated P(R^DGD). Moreover, the length of the PEG chain plays a major role in the ability to target the tumor. Hence, we can conclude that selectivity towards the tumor area of non-PEGylated and the PEGylated ICG-encapsulated P(R^DGD) NPs can be utilized for targeting to areas of angiogenesis, such as in the cases of tumors, wounds or cuts, etc.

Synthesis of NIR/ICG PEGylated poly(R^DGD) proteinoid NPs and their drug delivery towards mCherry-labeled 4T1 tumor.     

PEGylated insulin in PLGA microparticles. In vivo and in vitro analysis.

A novel controlled release formulation has been developed with PEGylated human insulin encapsulated in PLGA microspheres that produces multi-day release in vivo. The insulin is specifically PEGylated at the amino terminus of the B chain with a relatively low molecular weight PEG (5000 Da). Insulin with this modification retains full biological activity, but has a limited serum half-life, making encapsulation necessary for sustained release beyond a few hours. PEGylated insulin can be co-dissolved with PLGA in methylene chloride and microspheres made by a single o/w emulsion process. Insulin conformation and biological activity are preserved after PEGylation and PLGA encapsulation. The monolithic microspheres have inherently low burst release, an important safety feature for an extended release injectable insulin product. In PBS at 37 degrees C, formulations with a drug content of approximately 14% show very low (< 1%) initial release of insulin over one day and near zero order drug release after a lag of 3-4 days. In animal studies, PEG-insulin microspheres administered subcutaneously as a single injection produced < 1% release of insulin in the first day but then lowered the serum glucose levels of diabetic rats to values < 200 mg/dL for approximately 9 days. When doses were given at 7-day intervals, steady state drug levels were achieved after only 2 doses. PEG-insulin PLGA microparticles show promise as a once-weekly dosed, sustained release basal insulin formulation.     

Monolithic osmotic tablet system for nifedipine delivery.

The monolithic osmotic tablet system, which is composed of a monolithic tablet coated with cellulose acetate (CA) membrane drilled with two orifices on both side surfaces, has been described. The influences of tablet formulation variables including molecular weight (MW) and amount of polyethylene oxide (PEO), amount of potassium chloride (KCl), and amount of rice starch as well as nifedipine loading have been investigated. The optimal tablet formulation and the osmotic-suspending co-controlled delivery mechanisms have been proposed. Orifice size and membrane variables including nature and amount of plasticizers as well as thickness on drug release have also been studied. The in vitro release profiles of the optimal system have been evaluated in various release media and different agitation rates, and compared with commercialized conventional capsule and push-pull osmotic tablet. It was found that PEO with MW of 300000 g/mol was suitable to be thickening agent, both amount of KCl and amount of PEO had comparable and profoundly positive effects, while nifedipine loading had a strikingly negative influence on drug release. It could be found that the optimal orifice size was in the range of 0.25-1.41 mm. It has also been observed that hydrophilic plasticizer polyethylene glycol (PEG) improved drug release, whereas hydrophobic plasticizer triacetin depressed drug release when they were incorporated in CA membrane. The monolithic osmotic tablet system was found to be able to deliver nifedipine at the rate of approximate zero-order up to 24 h, independent of both environmental media and agitation rate, and substantially comparable with the push-pull osmotic tablet. The monolithic osmotic tablet system was simple to be prepared as exempting from push layer and simplifying in the orifice drilling compared with the push-pull osmotic tablet. The monolithic osmotic tablet system may be used in drug controlled delivery field, especially suitable for water-insoluble drugs.     

One-month sustained release microspheres of 125I-bovine calcitonin. In vitro-in vivo studies.

To obtain a 1-month release formulation of 125I-bovine calcitonin, microspheres were prepared with three different PLA copolymers, PLGA I (mol. wt. [MW]=30000), polyethyleneglycol (PEG)-PLGA (MW=34000) and PLGA II (MW=12000) using the double emulsion method. The release of 125I-bovine calcitonin was assayed in vitro using dialysis bags at 37 degrees C in isotonic phosphate buffer (pH 7.4). The in vitro release results indicated a very slow release rate for an optimal 1-month sustained release formulation. 125I-bovine calcitonin microspheres were administered under the skin on the back of Wistar rats and the radioactivity at the injection site was subsequently measured over a 4-week period. The in vitro and in vivo profiles were affected by the weight average molecular weight of the copolymers. The 125I-bovine calcitonin release rate was faster from microspheres prepared with PLGA II (MW=12000) than from microspheres prepared with higher molecular weight copolymers (PLGA I and PEG-PLGA). Microspheres prepared with PLGA II (MW=12000) release 100% of the dose in 1 month, in vivo release profiles presented two phases, during the first 2 weeks approximately 70% of the 125I-bovine calcitonin injected was released, followed by a second slower phase.     

A library of strictly linear poly(ethylene glycol)–poly(ethylene imine) diblock copolymers to perform structure–function relationship of non-viral gene carriers

A library of 39 strictly linear poly(ethylene glycol)–poly(ethylene imine) (PEG-PEI) diblock copolymers was synthesized for the delivery of plasmid DNA using PEG of 2, 5, or 10 kDa in combination with linear PEI with a molecular weight (MW) ranging from 1.5 to 10.8 kDa. In contrast to other approaches, the copolymers demonstrated a clear separation between the hydrophilic PEG and the nucleic acid condensing PEI moieties. Hence, the hypothesis was that PEG may not sterically counteract the interaction between the nucleic acid and PEI and that consequently, the copolymers are perfectly suited to build small and stable polyplexes. Analysis of the polyplexes revealed structure–function relationships and the general guideline was that the PEG domain had a greater influence on the physicochemical properties of the polyplexes than PEI. A PEG content higher than 50% led to small (< 150 nm), nearly neutral polyplexes with favorable stability. The transfection efficacy of these polyplexes was significantly reduced compared to the PEI homopolymer, but was restored by the application of the corresponding degradable copolymer, which involved a redox triggerable PEG domain. In conclusion, valuable design criteria for the optimization of gene delivery carriers, which is only possible through the screening of such a large library, were gained.     

Biodegradable star HPMA polymer-drug conjugates: Biodegradability, distribution and anti-tumor efficacy

Herein, new biodegradable star polymer-doxorubicin conjugates designed for passive tumor targeting were investigated, and their synthesis, physico-chemical characterization, drug release, biodegradation, biodistribution and in vivo anti-tumor efficacy are described. In the conjugates, the core formed by poly(amidoamine) (PAMAM) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing doxorubicin (Dox) attached by hydrazone bonds, which enabled intracellular pH-controlled drug release. The described synthesis facilitated the preparation of biodegradable polymer conjugates in a broad range of molecular weights (200-1000 g/mol) while still maintaining low polydispersity (~ 1.7). The polymer grafts were attached to the dendrimers through either stable amide bonds or enzymatically or reductively degradable spacers, which enabled intracellular degradation of the high-molecular-weight polymer carrier to excretable products. Biodegradability tests in suspensions of EL4 T-cell lymphoma cells showed that the rate of degradation was much faster for reductively degradable conjugates (close to completion within 24 h of incubation) than for conjugates linked via an enzymatically degradable oligopeptide GFLG sequence (slow degradation taking several days). This finding was likely due to the differences in steric hindrance in terms of the accessibility of the small molecule glutathione and the bulky enzyme cathepsin B to the polymer substrate. Regarding drug release, the conjugates were fairly stable in buffer at pH 7.4 (model of blood stream) but released doxorubicin under mild acidic conditions that model the tumor cell microenvironment. The star polymer-Dox conjugates exhibited significantly prolonged blood circulation and enhanced tumor accumulation in tumor-bearing mice, indicating the important role of the EPR effect in its anti-cancer activity. The star polymer conjugates showed prominently higher in vivo anti-tumor activities than the free drug or linear polymer conjugate when tested in mice bearing EL4 T-cell lymphoma, with a significant number of long-term surviving (LTS). Based on the results, we conclude that a M_w of HPMA copolymers of 200,000 to 600,000 g/mol is optimal for polymer carriers designed for the efficient passive targeting to solid tumors. In addition, an expressive therapy-dependent stimulation of the immune system was observed.