Synthesis and evaluation of a star amphiphilic block copolymer from poly(epsilon-caprolactone) and poly(ethylene glycol) as a potential drug delivery carrier

A star polymer composed of amphiphilic block copolymer arms has been synthesized and characterized. The core of the star polymer is polyamidoamine (PAMAM) dendrimer, the inner block in the arm is lipophilic poly(epsilon-caprolactone) (PCL), and the outer block in the arm is hydrophilic poly(ethylene glycol) (PEG). The star-PCL polymer was synthesized first by ring-opening polymerization of epsilon-caprolactone with a PAMAM-OH dendrimer as initiator. The PEG polymer was then attached to the PCL terminus by an ester-forming reaction. Characterization with SEC, (1)H NMR, FTIR, TGA, and DSC confirmed the star structure of the polymers. The micelle formation of the star copolymer (star-PCL-PEG) was studied by fluorescence spectroscopy. Hydrophobic dyes and drugs can be encapsulated in the micelles. A loading capacity of up to 22% (w/w) was achieved with etoposide, a hydrophobic anticancer drug. A cytotoxicity assay demonstrated that the star-PCL-PEG copolymer is nontoxic in cell culture. This type of block copolymer can be used as a drug delivery carrier.     

PAMAM-PEG-PAMAM: novel triblock copolymer as a biocompatible and efficient gene delivery carrier

A novel triblock copolymer, PAMAM-block-PEG-block-PAMAM was synthesized and applied as a gene carrier. PAMAM dendrimer is proven to be an efficient gene carrier itself, but it is associated with certain problems such as low water solubility and considerable cytotoxicity. Therefore, we introduced PEG to engineer a nontoxic and highly transfection efficient polymeric gene carrier because PEG is known to convey water-solubility and biocompatibility to the conjugated copolymer. This copolymer could achieve self-assembly with plasmid DNA, forming compact nanosized particles with a narrow size distribution. Fulfilling our expectations, the copolymer was found to form highly water-soluble polyplexes with plasmid DNA, showed little cytotoxicity despite its poor degradability, and finally achieved high transfection efficiency comparable to PEI in 293 cells. Consequently, these data show that an approach involving the introduction of PEG to create a tree-like cationic copolymer possesses a great potential for use in gene delivery systems.     

Dendronized chitosan derivative as a biocompatible gene delivery carrier

To improve the transfection efficiency of chitosan as a nonviral gene delivery vector, a dendronized chitosan derivative was prepared by a copper-catalyzed azide alkyne cyclization reaction of propargyl focal point poly(amidoamine) dendron with 6-azido-6-deoxy-chitosan. Its structure was characterized by (1)H NMR and FTIR analyses and its buffering capacity was evaluated by acid-base titration. In particular, its complexation with plasmid DNA was investigated by agarose gel electrophoresis, zeta potential, and particle size analyses as well as transmission electron microscopy observation. Compared to unmodified chitosan, such a chitosan derivative has better water solubility and buffering capacity. Compared to commonly used polyethyleneimine (PEI, 25 kDa), it could exhibit enhanced transfection efficiency in some cases and lower cell toxicity, as confirmed by in vitro transfection and cytotoxicity tests in human kidney 293T and human nasopharyngeal carcinoma CNE2 cell lines. In addition, the effect of serum on its transfection efficiency was also studied.     

Characterization of hydrophobic anti-cancer drug-loaded amphiphilic peptides as a gene carrier

An amphiphilic peptide with a 3-arginine stretch and a 6-valine stretch was evaluated as a gene carrier. The short amphiphilic peptide, R3V6, not only formed micelles in aqueous solution, but was also able to deliver plasmid DNA (pDNA) into cells without toxicity. In this research, various amphiphilic peptides were synthesized with a 3-arginine stretch and a 6-valine, -alanine, -leucine, or -phenylalanine stretch. In vitro transfection assays in human embryonic kidney 293 cells showed that R3V6 and R3L6 peptides had higher transfection efficiencies than R3A6, R3F6, and poly-L-lysine (PLL). Since the peptide micelles had hydrophobic cores, a hydrophobic anti-cancer drug, bis-chloronitrosourea (BCNU),was able to be loaded into the cores of the micelles. The incorporation of the hydrophobic drug into the cores of the peptide micelles may stabilize the micelle structure and increase the transfection efficiency. The in vitro transfection assay with BCNU-loaded R3V6 (R3V6-BCNU) or R3L6 (R3L6-BCNU) showed that the BCNU-loaded peptide micelles had a higher transfection efficiency than the peptide micelles without BCNU. R3V6-BCNU and R3L6-BCNU had the highest transfection at a 0.8:1 weight ratio (BCNU:R3V6) and a 1.2:1 weight ratio (BCNU:R3L6), respectively. Furthermore, compared to simple diffusion, a more efficient delivery of the drug into cells may be facilitated by endocytosis of the micelles. R3L6-BCNU and R3V6-BCNU had higher cell toxicity to cells than BCNU alone. Therefore, the R3V6- and R3L6-BCNU may be useful for drug and gene combination cancer therapy.     

An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems

Intelligent gene delivery systems based on physiologically triggered reversible shielding technology have evinced enormous interest due to their potential in vivo applications. In the present work, an acid-labile block copolymer consisting of poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacrylate) segments connected through a cyclic ortho ester linkage (PEG- a-PDMAEMA) was synthesized by atom transfer radical polymerization of DMAEMA using a PEG macroinitiator with an acid-cleavable end group. PEG- a-PDMAEMA condensed with plasmid DNA formed polyplex nanoparticles with an acid-triggered reversible PEG shield. The pH-dependent shielding/deshielding effect of PEG chains on the polyplex particles were evaluated by zeta potential and size measurements. At pH 7.4, polyplexes generated from PEG- a-PDMAEMA exhibited smaller particle size, lower surface charge, reduced interaction with erythrocytes, and less cytotoxicity compared to PDMAEMA-derived polyplexes. At pH 5.0, zeta potential of polyplexes formed from PEG- a-PDMAEMA increased, leveled up after 2 h of incubation and gradual aggregation occurred in the presence of bovine serum albumin (BSA). In contrast, the stably shielded polyplexes formed by DNA and an acid-stable block copolymer, PEG- b-PDMAEMA, did not change in size and zeta potential in 6 h. In vitro transfection efficiency of the acid-labile copolymer greatly increased after 6 h incubation at pH 5.0, approaching the same level of PDMAEMA, whereas there was only slight increase in efficiency for the stable copolymer, PEG- b-PDMAEMA.     

Biodegradable amphiphilic copolymer containing nucleobase: synthesis, self-assembly in aqueous solutions, and potential use in controlled drug delivery

Biodegradable nucleobase-grafted amphiphilic copolymer, the methoxyl poly (ethylene glycol)-b-poly (L-lactide-co-2-methyl-2(3-(2,3-dihydroxylpropylthio) propyloxycarbonyl)-propylene carbonate/1-carboxymethylthymine) (mPEG-b- P(LA-co-MPT)), was synthesized. (1)H NMR titration and FT-IR spectroscopy indicated that the hydrogen-bonding could be formed between mPEG-b-P(LA-co-MPT) and 9-hexadecyladenine (A-C16). The hydrophobic microenvironment of the amphiphilic copolymer can protect the complementary multiple hydrogen bonds between mPEG-b-P(LA-co-MPT) and A-C16 from water effectively. The addition of A-C16 not only lowered the critical aggregation concentration (CAC) of mPEG-b-P(LA-co-MPT)/A-C16 nanoparticles (NPs) in aqueous solution but also induced different morphologies, which can be observed by transmission electron microscopy (TEM). Meanwhile, dynamic light scattering (DLS) and turbidometry was utilized to evaluate the effect of temperature and pH change on the stability of mPEG-b-P(LA-co-MPT)/A-C16 NPs. Cytotoxicity evaluation showed good biocompatibility of the mPEG-b-P(LA-co-MPT)/A-C16 NPs. The in vitro drug release profile showed that with the increase of A-C16 content, the doxorubiucin (DOX) release at pH 7.4 decreased, while the faster release rate was observed with the addition of A-C16 with a pH of 5.0. Importantly, DOX-loaded NPs exerted comparable cytotoxicity against MDA-MB-231 cells. This work provided a new method to stabilize NP structure using hydrogen-bonds and would have the potential to be applied in controlled drug delivery.     

Bioreducible micelles self-assembled from amphiphilic hyperbranched multiarm copolymer for glutathione-mediated intracellular drug delivery

A new type of biodegradable micelles for glutathione-mediated intracellular drug delivery was developed on the basis of an amphiphilic hyperbranched multiarm copolymer (H40-star-PLA-SS-PEP) with disulfide linkages between the hydrophobic polyester core and hydrophilic polyphosphate arms. The resulting copolymers were characterized by nuclear magnetic resonance (NMR), Fourier transformed infrared (FTIR), gel permeation chromatography (GPC), and differential scanning calorimeter (DSC) techniques. Benefiting from amphiphilic structure, H40-star-PLA-SS-PEP was able to self-assemble into micelles in aqueous solution with an average diameter of 70 nm. Moreover, the hydrophilic polyphosphate shell of these micelles could be detached under reduction-stimulus by in vitro evaluation, which resulted in a rapid drug release due to the destruction of micelle structure. The glutathione-mediated intracellular drug delivery was investigated against a Hela human cervical carcinoma cell line. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements demonstrated that H40-star-PLA-SS-PEP micelles exhibited a faster drug release in glutathione monoester (GSH-OEt) pretreated Hela cells than that in the nonpretreated cells. Cytotoxicity assay of the doxorubicin-loaded (DOX-loaded) micelles indicated the higher cellular proliferation inhibition against 10 mM of GSH-OEt pretreated Hela cells than that of the nonpretreated ones. As expected, the DOX-loaded micelles showed lower inhibition against 0.1 mM of buthionine sulfoximine (BSO) pretreated Hela cells. These reduction-responsive and biodegradable micelles show a potential to improve the antitumor efficacy of hydrophobic chemotherapeutic drugs.     

Folate-functionalized unimolecular micelles based on a degradable amphiphilic dendrimer-like star polymer for cancer cell-targeted drug delivery

A folate-functionalized degradable amphiphilic dendrimer-like star polymer (FA-DLSP) with a well-defined poly(L-lactide) (PLLA) star polymer core and six hydrophilic polyester dendrons based on 2,2-bis(hydroxymethyl) propionic acid was successfully synthesized to be used as a nanoscale carrier for cancer cell-targeted drug delivery. This FA-DLSP hybrid formed unimolecular micelles in the aqueous solution with a mean particle size of ca. 15 nm as determined by dynamic light scattering and transmission electron microscopy. To study the feasibility of FA-DLSP micelles as a potential nanocarrier for targeted drug delivery, we encapsulated a hydrophobic anticancer drug, doxorubicin (DOX), in the hydrophobic core, and the loading content was determined by UV-vis analysis to be 4 wt %. The DOX-loaded FA-DLSP micelles demonstrated a sustained release of DOX due to the hydrophobic interaction between the polymer core and the drug molecules. The hydrolytic degradation in vitro was monitored by weight loss and proton nuclear magnetic resonance spectroscopy to gain insight into the degradation mechanism of the FA-DLSP micelles. It was found that the degradation was pH-dependent and started from the hydrophilic shell gradually to the hydrophobic core. Flow cytometry and confocal microscope studies revealed that the cellular binding of the FA-DLSP hybrid against human KB cells with overexpressed folate-receptors was about twice that of the neat DLSP (without FA). The in vitro cellular cytotoxicity indicated that the FA-DLSP micelles (without DOX) had good biocompatibility with KB cells, whereas DOX-loaded micelles exhibited a similar degree of cytotoxicity against KB cells as that of free DOX. These results clearly showed that the FA-DLSP unimolecular micelles could be a promising nanosize anticancer drug carrier with excellent targeting property.     

PEG-SS-PPS: reduction-sensitive disulfide block copolymer vesicles for intracellular drug delivery

Under appropriate conditions, block copolymeric macroamphiphiles will self-assemble in water to form vesicles, referred to as polymersomes. We report here polymersomes that can protect biomolecules in the extracellular environment, are taken up by endocytosis, and then suddenly burst within the early endosome, releasing their contents prior to exposure to the harsh conditions encountered after lysosomal fusion. Specifically, block copolymers of the hydrophile poly(ethylene glycol) (PEG) and the hydrophobe poly(propylene sulfide) (PPS) were synthesized with an intervening disulfide, PEG17-SS-PPS30. Polymersomes formed from this block copolymer were demonstrated to disrupt in the presence of intracellular concentrations of cysteine. In cellular experiments, uptake, disruption, and release were observed within 10 min of exposure to cells, well within the time frame of the early endosome of endolysosomal processing. This system may be useful in cytoplasmic delivery of biomolecular drugs such as peptides, proteins, oligonucleotides, and DNA.     

Synthesis and characterization of amphiphilic glycidol-chitosan-deoxycholic acid nanoparticles as a drug carrier for doxorubicin

Novel amphiphilic chitosan derivatives (glycidol-chitosan-deoxycholic acid, G-CS-DCA) were synthesized by grafting hydrophobic moieties, deoxycholic acid (DCA), and hydrophilic moieties, glycidol, with the purpose of preparing carriers for poorly soluble drugs. Based on self-assembly, G-CS-DCA can form nanoparticles with size ranging from 160 to 210 nm, and G-CS-DCA nanoparticles maintained stable structure for about 3 months when stored in PBS (pH 7.4) at room temperature. The critical aggregation concentration decreased from 0.043 mg/mL to 0.013 mg/mL with the increase of degree of substitution (DS) of DCA. Doxorubicin (DOX) could be easily encapsulated into G-CS-DCA nanoparticles and keep a sustained release manner without burst release when exposed to PBS (pH 7.4) at 37 °C. Antitumor efficacy results showed that DOX-G-CS-DCA have significant antitumor activity when MCF-7 cells were incubated with different concentration of DOX-G-CS-DCA nanoparticles. The fluorescence imaging results indicated DOX-G-CS-DCA nanoparticles could easily be uptaken by MCF-7 cells. These results suggested that G-CS-DCA nanoparticles may be a promising carrier for DOX delivery in cancer therapy.     

Polymeric micelles with water-insoluble drug as hydrophobic moiety for drug delivery

The hydrophobic block of polymeric micelles formed by amphiphilic copolymers has no direct therapeutical effect, and the metabolites of these hydrophobic segments might lead to some unexpected side effects. Here the hydrophobic core of polymeric micelles is replaced by highly water-insoluble drugs themselves, forming a new micellar drug delivery system. By grafting hydrophobic drugs of paclitaxel (PTX) onto the surface of hydrophilic hyperbranched poly(ether-ester) (HPEE), we constructed an amphiphilic copolymer (HPEE-PTX). HPEE-PTX could self-assemble into micellar nanoparticles in aqueous solution with tunable drug contents from 4.1 to 10.7%. Moreover, the hydrolysis of HPEE-PTX in serum resulted in the cumulative release of PTX. In vivo evaluation indicated that the dosage toleration of PTX in mice had been improved greatly and HPEE-PTX micellar nanoparticles could be used as an efficient prodrug with satisfactory therapeutical effect. We believe that most of the lipophilic drugs could improve their characters through this strategy.     

Synthesis of amphiphilic star block copolymers and their evaluation as transdermal carriers

Amphiphilic star polymers offer substantial promise for a range of drug delivery applications owing to their ability to encapsulate guest molecules. One appealing but underexplored application is transdermal drug delivery using star block copolymer reverse micelles as an alternative to the more common oral and intravenous routes. We prepared 6- and 12-arm amphiphilic star copolymers via atom transfer radical polymerization (ATRP) of sequential blocks of polar oligo (ethylene glycol)methacrylate and nonpolar lauryl methacrylate from brominated dendritic macroinitiators based on 2,2-bis(hydroxymethyl) propionic acid. These star block copolymers demonstrate the ability to encapsulate polar dyes such as rhodamine B and FITC-BSA in nonpolar media via UV/vis spectroscopic studies and exhibit substantially improved encapsulation efficiencies, relative to self-assembled "1-arm" linear block copolymer analogs. Furthermore, their transdermal carrier capabilities were demonstrated in multiple dye diffusion studies using porcine skin, verifying penetration of the carriers into the stratum corneum.     

Plant protein-based hydrophobic fine and ultrafine carrier particles in drug delivery systems

For thousands of years, plants and their products have been used as the mainstay of medicinal therapy. In recent years, besides attempts to isolate the active ingredients of medicinal plants, other new applications of plant products, such as their use to prepare drug delivery vehicles, have been discovered. Nanobiotechnology is a branch of pharmacology that can provide new approaches for drug delivery by the preparation of biocompatible carrier nanoparticles (NPs). In this article, we review recent studies with four important plant proteins that have been used as carriers for targeted delivery of drugs and genes. Zein is a water-insoluble protein from maize; Gliadin is a 70% alcohol-soluble protein from wheat and corn; legumin is a casein-like protein from leguminous seeds such as peas; lectins are glycoproteins naturally occurring in many plants that recognize specific carbohydrate residues. NPs formed from these proteins show good biocompatibility, possess the ability to enhance solubility, and provide sustained release of drugs and reduce their toxicity and side effects. The effects of preparation methods on the size and loading capacity of these NPs are also described in this review.     

Synthesis and characterizations of amphiphilic poly(L-lactide)-grafted chondroitin sulfate copolymer and its application as drug carrier

In this investigation, new biodegradable brush-like amphiphilic copolymers were synthesized by ring opening polymerization. Poly(L-lactide) (PLLA) was grafted onto chondroitin sulfate (CS), which is one of the physiologically significant specific glycosaminoglycans (GAGs), using a tin octanoate [Sn(Oct)2] catalyst in DMSO. The hydroxyl groups of the chondroitin sulfate were used as initiating groups. These functional groups enable specific mucoadhesion or receptor recognition. The degree of substitution (DS), the degree of polymerization (DP) and the chondroitin sulfate content (from 1.1 to 15.4%) were analyzed by 1H NMR. The characteristics of these grafted copolymers, including the structure, the thermal properties and biodegradability, etc., were examined with respect to CS content. Meanwhile, the amphiphilic core (PLLA)-corona (CS) nanoparticles, with size smaller than 200 nm, was examined by dynamic light scattering (DLS). Zeta potential analysis exhibited the value in the range -18.3 to -49.4 mV. The morphologies of the nanoparticles were observed by field-emission scanning electron microscopy (FE-SEM). The nanoparticles with lower cytotoxicity were examined by MTT assay. Furthermore, the in vitro BSA release kinetics of those CSn-PLLA nanoparticles was also determined in this study.     

Evaluation of amylose used as a drug delivery carrier

The enzyme-dependent conjugates of indomethacin and amylose (Am-IND) were synthesized at room temperature using N,N′-dicyclohexylcarbodiimide (DCC) as a coupling agent and 4-(N,N′-dimethylamino) pyridine (DMAP) as a catalyst. Their structures were characterized by FTIR and ¹H NMR analyses, and the results indicated that the IND residues were conjugated with amylose backbones through ester bonds. For the conjugate with a lower IND content, the better water absorption property was advantageous for enzymes diffusing into the swollen conjugate, resulting in biodegradation of the conjugates and release of IND. In vitro biodegradation evaluation indicated that the Am-IND conjugates were biodegraded in the simulated media of the intestines. In vitro drug release experiments showed that the Am-IND conjugates exhibited a sustained release behavior in the simulated media of the intestines, while IND was hardly released in the simulated gastric fluid. These features provide a great opportunity to use the conjugates as a prodrug for intestinally targeted and controlled release of IND through oral administration. This study may lead to the development of effective methods for utilizing amylose as a new drug delivery carrier.     

Fully acid-degradable biocompatible polyacetal microparticles for drug delivery

A library of polyurethanes and polyureas with different hydrophobicities containing the same acid-degradable dimethyl ketal moiety embedded in the polymer main chain have been prepared. All polymers were synthesized using an AA-BB type step-growth polymerization by reaction of bis(p-nitrophenyl carbamate/carbonate) or diisocyanate monomers with an acid-degradable, ketal-containing diamine. These polymers were designed to hydrolyze at different rates in mildly acidic conditions as a function of their hydrophobicity to afford small molecules only with no polymeric byproduct. The library of polymers was screened for the formation of microparticles using a double emulsion technique. The microparticles that were obtained degraded significantly faster at acidic pH (5.0) than at physiological pH (7.4) with degradation kinetics related to the hydrophobicity of the starting polymer. In vitro studies demonstrated the ability of the FITC-BSA loaded microparticles to be phagocytosed by macrophages resulting in a 10-fold increase in the protein uptake compared to a free protein control; in addition, the microparticles were found to be nontoxic at the concentrations tested of up to 1 mg/mL. The ease of preparation of the polymers coupled with the ability to tune their hydrophobicity and the high acid sensitivity of the microparticles identify this new class of materials as promising candidates for the delivery of bioactive materials.     

Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

Viral-mediated gene delivery has been explored for the treatment and protection of cardiomyocytes, but so far there is only one report using cationic polymer for gene delivery to cardiomyocytes in spite of many advantages of polymer-mediated gene delivery. In this study, a cationic poly(beta-amino ester) (PDMA) with a degradable backbone and cleavable side chains was synthesized by Michael addition reaction. The toxicity of PDMA to neonatal mouse cardiomyocytes (NMCMs) was significantly lower than that of polyethyleneimine (PEI). PDMA formed stable polyplexes with pEGFP. The dissociation of the polyplexes could be triggered by PDMA degradation, and the dissociation time was tunable via the polymer/pEGFP ratio. In vitro transfection showed that PDMA was an effective and low toxic gene delivery carrier for NMCMs. The PDMA/pEGFP polyplexes transfected EGFP gene to NMCMs with about 28% efficiency and caused little death. In contrast, a significant portion of cardiomyocytes cultured with PEI/pEGFP died.     

Mesoscale simulation on patterned nanotube model for amphiphilic block copolymer

Self-assembly of AB diblock copolymer confined in concentric-cylindrical nanopores was studied using MesoDyn simulation. Our calculation shows that in this confined geometry a zoo of exotic structures can be formed. These structures include bicontinuous phases like carbon nanotube, imperfect single helixes and double helixes. Moreover, the dependence of the chain conformation on the volume fraction, concentration, the interactions between blocks and the diameter of the cylindrical pore are investigated. The results of these simulations can be used to predict the diblock copolymer morphologies confined in concentric-cylindrical nanopores and should be helpful in designing polymeric nanomaterials in the future.     

Synthesis of well-defined amphiphilic block copolymers having phospholipid polymer sequences as a novel biocompatible polymer micelle reagent

To realize safer and effective drug administration, novel well-defined and biocompatible amphiphilic block copolymers containing phospholipid polymer sequences were synthesized. At first, the homopolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) was synthesized in water by reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. The "living" polymerization was confirmed by the fact that the number-average molecular weight increased linearly with monomer conversion while the molecular weight distribution remained narrow independent of the conversion. The poly(MPC) thus prepared is end-capped with a dithioester moiety. Using the dithioester-capped poly(MPC) as a macro chain transfer agent, AB diblock copolymers of MPC and n-butyl methacrylate (BMA) were synthesized. Associative properties of the amphiphilic block copolymer (pMPC(m)-BMA(n)) with varying poly(BMA) block lengths were investigated using NMR, fluorescence probe, static light scattering (SLS), and quasi-elastic light scattering (QELS) techniques. Proton NMR data in D2O indicated highly restricted motions of the n-butyl moieties, arising from hydrophobic associations of poly(BMA) blocks. Fluorescence spectra of N-phenyl-1-naphthylamine indicated that the probes were solubilized in the polymer micelles in water. The formation of polymer micelles comprising a core with poly(BMA) blocks and shell with hydrophilic poly(MPC) blocks was suggested by SLS and QELS data. The size and mass of the micelle increased with increasing poly(BMA) block length. With an expectation of a pharmaceutical application of pMPC(m)-BMA(n), solubilization of a poorly water-soluble anticancer agent, paclitaxel (PTX), was investigated. PTX dissolved well in aqueous solutions of pMPC(m)-BMA(n) as compared with pure water, implying that PTX is incorporated into the hydrophobic core of the polymer micelle. Since excellent biocompatible poly(MPC) sequences form an outer shell of the micelle, pMPC(m)-BMA(n) may find application as a promising reagent to make a good formulation with a hydrophobic drug.