Folate-conjugated amphiphilic hyperbranched block copolymers based on Boltorn® H40, poly(l-lactide) and poly(ethylene glycol) for tumor-targeted drug delivery

Folate-conjugated amphiphilic hyperbranched block copolymer (H40–PLA-b-MPEG/PEG–FA) with a dendritic Boltorn^® H40 core, a hydrophobic poly(l-lactide) (PLA) inner shell and a hydrophilic methoxy poly(ethylene glycol) (MPEG) and folate-conjugated poly(ethylene glycol) (PEG–FA) outer shell was synthesized as a carrier for tumor-targeted drug delivery. The block copolymer was characterized using ¹H NMR and gel permeation chromatography (GPC) analysis. Due to its core–shell structure, this block polymer forms unimolecular micelles in aqueous solutions. The micellar properties of H40–PLA-b-MPEG/PEG–FA block copolymer were extensively studied by dynamic light scattering (DLS), fluorescence spectroscopy, and transmission electron microscopy (TEM). An anticancer drug, doxorubicin in the free base form (DOX) was encapsulated into H40–PLA-b-MPEG/PEG–FA micelles. The DOX-loaded micelles provided an initial burst release (up to 4 h) followed by a sustained release of the entrapped DOX over a period of about 40 h. Cellular uptake of the DOX-loaded H40–PLA-b-MPEG/PEG–FA micelles was found to be higher than that of the DOX-loaded H40–PLA-b-MPEG micelles because of the folate-receptor-mediated endocytosis, thereby providing higher cytotoxicity against the 4T1 mouse mammary carcinoma cell line. In vitro degradation studies revealed that the H40–PLA-b-MPEG/PEG–FA block copolymer hydrolytically degraded into polymer fragments within six weeks. These results indicated that the micelles prepared from the H40–PLA-b-MPEG/PEG–FA block copolymer have great potential as tumor-targeted drug delivery nanocarriers.     

Folic acid conjugated glycol chitosan micelles for targeted delivery of doxorubicin: preparation and preliminary evaluation in vitro

For folate receptor (FR) targeted anticancer therapy, novel folic acid (FA) conjugated cholesterol-modified glycol chitosan (FCHGC) micelles were synthesized and characterized by ¹H NMR, dynamic light scattering, transmission electron microscopy, and fluorescence spectroscopy. The degree of substitution was 1.4 FA groups and 7.7 cholesterol groups per 100 sugar residues of glycol chitosan. The critical aggregation concentration of FCHGC micelles in aqueous solution was 0.0169?mg/ml. The doxorubicin (DOX)-loaded FCHGC (DFCHGC) micelles were prepared by an emulsion/solvent evaporation method. The DFCHGC micelles were almost spherical in shape and their size increased from 282 to 320?nm with the DOX-loading content increasing from 4.53 to 11.4%. DOX released from DOX-loaded micelles displayed sustained release behavior. The targeted micelles encapsulated DOX showed significantly greater cytotoxicity against FR-positive HeLa cells than the nontargeted DOX-loaded micelles and free DOX. These results suggested that FCHGC micelles could be a potential carrier for targeted drug delivery.     

Folate-functionalized polymeric micelle as hepatic carcinoma-targeted,MRI-ultrasensitive delivery system of antitumor drugs

Targeted delivery is a highly desirable strategy to improve the diagnostic imaging and therapeutic outcome because of enhanced efficacy and reduced toxicity. In the current research, anticancer drug doxorubicin (DOX) and contrast agent for magnetic resonance imaging (MRI), herein superparamagnetic ion oxide Fe(3)O(4) (SPIO), were accommodated in the core of micelles self-assembled from amphiphilic block copolymer of poly(ethylene glycol) (PEG) and poly(varepsilon-caprolactone) (PCL) with a targeting ligand (folate) attached to the distal ends of PEG (Folate-PEG-PCL). The in vitro tumor cell targeting efficacy of these folate functionalized and DOX/SPIO-loaded micelles (Folate-SPIO-DOX-Micelles) was evaluated upon observing cellular uptake of micelles by human hepatic carcinoma cells (Bel 7402 cells) which overexpresses surface receptors for folic acid. In the Prussian blue staining experiments, cells incubated with Folate-SPIO-DOX-Micelles showed much higher intracellular iron density than the cells incubated with the folate-free SPIO-DOX-Micelles. According to the flow cytometry data, cellular DOX uptake observed for the folate targeting micelle was about 2.5 fold higher than that for the non-targeting group. Furthermore, MTT assay showed that Folate-SPIO-DOX-Micelles effectively inhibited cell proliferation, while the folate-free SPIO-DOX-Micelles did not show the same feat at comparable DOX concentrations. The potential of Folate-SPIO-DOX-Micelle as a novel MRI-visible nanomedicine platform was assessed with a 1.5 T clinical MRI scanner. The acquired MRI T (2) signal intensity of cells treated with the folate targeting micelles decreased significantly. By contrast, T (2) signal did not show obvious decrease for cells treated with the folate-free micelles. Our results indicate that the multifunctional polymeric micelles, Folate-SPIO-DOX-Micelles, have better targeting tropism to the hepatic carcinoma cells in vitro than their non-targeting counterparts, and the cell targeting events of micelles can be monitored using a clinical MRI scanner.     

Incorporation and in vitro release of doxorubicin in thermally sensitive micelles made from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glycolide) with varying compositions

Thermally sensitive block copolymers, poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide)-b-poly(d,l-lactide-co-glycolide) [P(NIPAAm-co-DMAAm)-b-poly(D,L-lactide-co-glycolide) (PLGA)] with different compositions and lengths of PLGA block are synthesized and utilized to fabricate micelles containing doxorubicin (DOX), a model anticancer drug, by a membrane dialysis method for targeted anticancer drug delivery. The critical association concentration (CAC) of the polymers ranges from 4.0 to 25.0 mg/L. An increased length of core-forming block PLGA leads to a decrease in the CAC. The clearly defined core-shell structure of micelles is proved by 1H-NMR analyses of the micelles in CDCl3 and D2O. The morphology of the micelles is analyzed by transmission electron microscopy, showing a spherical structure of both blank and drug-loaded micelles. The results obtained from dynamic light scattering show that the blank and drug-loaded micelles have an average size below 200 nm. The lower critical solution temperature (LCST) of the micelles made from the various polymers is similar, around 39 degrees C in phophate-buffered solution (PBS). The presence of serum in PBS does not alter the LCST significantly. The drug loading capacity varies depending on the PLGA block. The polymers are degradable, and the degradation of PLGA-based polymers is faster than that of poly(lactide) (PLA)-based polymer. The DOX-loaded micelles are stable in PBS containing serum at 37 degrees C but deform at 39.5 degrees C above the normal body temperature, thus triggering DOX release. It is revealed by confocal laser scanning microscopy that free DOX molecules enter cell nuclei very fast and DOX-loaded micelles accumulate mostly in cytoplasm after endocytosis. At a temperature above the LCST, more DOX molecules release from the micelles and enter the nuclei as compared to the temperature below the LCST. DOX-loaded micelles show greater cytotoxicity at a temperature above the LCST. The P(NIPAAm-co-DMAAm)-b-PLGA micelles developed may be a good carrier for anticancer drug delivery.     

The role of non-covalent interactions in anticancer drug loading and kinetic stability of polymeric micelles

A new series of acid- and urea-functionalized polycarbonate block copolymers were synthesized via organocatalytic living ring-opening polymerization using methoxy poly(ethylene glycol) (PEG) as a macroinitiator to form micelles as drug delivery carriers. The micelles were characterized for critical micelle concentration, particle size and size distribution, kinetic stability and loading capacity for a model anticancer drug, doxorubicin (DOX) having an amine group. The acid/urea groups were placed in block forms (i.e. acid as the middle block or the end block) or randomly distributed in the polycarbonate block to investigate molecular structure effect. The micelles formed from the polymers in both random and block forms provided high drug loading capacity due to strong ionic interaction between the acid in the polymer and the amine in DOX. However, the polymers with acid and urea groups placed in the block forms formed micelles with wider size distribution (two size populations), and their DOX-loaded micelles were less stable. The number of acid/urea groups in the random form was further varied from 5 to 8, 13 and 19 to study its effects on self-assembly behaviors and DOX loading. An increased number of acid/urea groups yielded DOX-loaded micelles with smaller size and enhanced kinetic stability because of improved inter-molecular polycarbonate-polycarbonate (urea-urea and urea-acid) hydrogen-bonding and polycarbonate-DOX (acid-amine) ionic interactions. However, when the number of acid/urea groups was 13 or higher, micelles aggregated in a serum-containing medium, and freeze-dried DOX-loaded micelles were unable to re-disperse in an aqueous solution. Among all the polymers synthesized in this study, 1b with 8 acid/urea groups in the random form had the optimum properties. In vitro release studies showed that DOX release from 1b micelles was sustained over 7 h without significant initial burst release. MTT assays demonstrated that the polymer was not toxic towards HepG2 and HEK293 cells. Importantly, DOX-loaded micelles were potent against HepG2 cells with IC₅₀ of 0.26 mg/L, comparable to that of free DOX (IC₅₀: 0.20 mg/L). In addition, DOX-loaded 1b micelles yielded lower DOX content in the heart tissue of the tested mice as compared to free DOX formulation after i.v. injection. These findings signify that 1b micelles may be a promising carrier for delivery of anticancer drugs that contain amine groups.     

Doxorubicin-Loaded Nanosized Micelles of a Star-Shaped Poly(ε-Caprolactone)-Polyphosphoester Block Co-polymer for Treatment of Human Breast Cancer

Star-shaped co-polymers based on the backbone of poly(ε-caprolactone) were synthesized by a ring-opening reaction using pentaerythritol as initiator and Sn(Oct)₂ as catalyst. The star-shaped poly(ε-caprolactone) polymer was then chain extended with a terminal block of poly(ethyl ethylene phosphate) to form a copolymer, poly(ε-caprolactone)-poly(ethyl ethylene phosphate), when using the cyclic ethyl ethylene phosphate monomer. The amphiphilic block co-polymers can self-assemble into nanoscopic micelles with a mean diameter of 150 nm and a spherical shape. Additionally, the prepared micelles did not induce hemolysis and nitric oxide production in vitro based on nitric oxide, hemolytic tests and MTT assays. The hydrophobic micellar cores encapsulated doxorubicin (DOX) in an aqueous solution with a loading efficiency of 55.2%. The in vitro release of DOX from DOX-loaded micelles was pH dependent. DOX-loaded micelles present significantly enhanced cytotoxicity to both MCF-7/drug-sensitive and MCF-7/drug-resistant cells after second incubation. Moreover, results of confocal microscopy and flow cytometry of DOX-loaded micelles demonstrate the feasibility of this delivery system for effective therapy of drug-resistant tumours.     

Amphiphilic multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery

Folate-conjugated unimolecular micelles based on amphiphilic hyperbranched block copolymer, Boltorn^® H40-poly(l-aspartate-doxorubicin)-b-poly(ethylene glycol)/FA-conjugated poly(ethylene glycol) (H40-P(LA-DOX)-b-PEG-OH/FA), were synthesized as a carrier for tumor-targeted drug delivery. The anticancer drug DOX was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms by pH-sensitive hydrazone linkage. The size of the unimolecular micelles was determined as 17–36 and 10–20 nm by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The release profiles of the DOX from the H40-P(LA-DOX)-b-PEG-OH/FA micelles showed a strong dependence on the environmental pH values. The DOX release rate increased in the acidic medium due to the acid-cleavable hydrazone linkage between the DOX and micelles. Cellular uptake of the H40-P(LA-DOX)-b-PEG-OH/FA micelles was found to be higher than that of the H40-P(LA-DOX)-b-PEG-OH micelles because of the folate-receptor-mediated endocytosis, thereby providing higher cytotoxicity against the 4T1 mouse mammary carcinoma cell line. Degradation studies showed that the H40-P(LA-DOX)-b-PEG-OH/FA copolymer hydrolytically degraded into polymer fragments within six weeks. These results suggest that H40-P(LA-DOX)-b-PEG-OH/FA micelles could be a promising nanocarrier with excellent in vivo stability for targeting the drugs to cancer cells and releasing the drug molecules inside the cells by sensing the acidic environment of the endosomal compartments.     

Polymeric topology and composition constrained polyether–polyester micelles for directional antitumor drug delivery

Amphiphilic linear and dumbbell-shaped poly(ethylene glycol)–poly(lactide-co-glycolide) (PEG–PLGA) copolymers were simply synthesized by the ring-opening polymerization of lactide and glycolide using PEG or tetrahydroxyl-functionalized PEG as the macroinitiator and stannous octoate as the catalyst. The copolymers spontaneously self-assembled into spherical micelles in phosphate-buffered saline at pH 7.4. The self-assembly behavior was dependent on both the polymeric topology and composition. Doxorubicin (DOX), an anthracycline antitumor drug, was loaded into micelles through nanoprecipitation. The in vitro release behavior could be adjusted by regulating the topology or composition of the copolymer, or the pH of the release medium. The effective intracellular DOX release from DOX-loaded micelles was confirmed by confocal laser scanning microscopy and flow cytometry in vitro. DOX-loaded micelles displayed great cellular proliferation inhibition efficacies after incubation for 24, 48 or 72 h. The hemolysis ratio of DOX was significantly reduced by the presence of copolymer. These properties indicated that the micelles derived from linear or dumbbell-shaped copolymers were promising candidates as smart antitumor drug carriers for malignancy therapy.     

Self-assembled pH-responsive hyaluronic acid–g-poly(l-histidine) copolymer micelles for targeted intracellular delivery of doxorubicin

Hyaluronic acid (HA) was conjugated with hydrophobic poly(l-histidine) (PHis) to prepare a pH-responsive and tumor-targeted copolymer, hyaluronic acid–g-poly(l-histidine) (HA-PHis), for use as a carrier for anti-cancer drugs. The effect of the degree of substitution (DS) on the pH-responsive behaviour of HA-PHis copolymer micelles was confirmed by studies of particles of different sizes. In vitro drug release studies demonstrated that doxorubicin (DOX) was released from HA-PHis micelles in a pH-dependent manner. In vitro cytotoxicity assays showed that all the blank micelles were nontoxic. However, MTT assay against Michigan Cancer Foundation-7 (MCF-7) cells (overexpressed CD44 receptors) showed that DOX-loaded micelles with a low PHis DS were highly cytotoxic. Cellular uptake experiments revealed that these pH-responsive HA-PHis micelles taken up in great amounts by receptor-mediated endocytosis and DOX were efficiently delivered into cytosol. Moreover, micelles with the lowest DS exhibited the highest degree of cellular uptake, which indicated that the micelles were internalized into cells via CD44 receptor-mediated endocytosis and the carboxylic groups of HA are the active binding sites for CD44 receptors. Endocytosis inhibition experiments and confocal images demonstrated that HA-PHis micelles were internalized into cells mainly via clathrin-mediated endocytosis and delivered to lysosomes, triggering release of DOX into the cytoplasm. These results confirm that the biocompatible pH-responsive HA-PHis micelles are a promising nanosystem for the intracellular targeted delivery of DOX.     

Anticancer Activity of Released Doxorubicin from a Folate-Mediated Polyelectrolyte Complex

Folic acid (FA) was selected to link with macromolecules for the selective and specific delivery of doxorubicin (DOX) to folate receptor (FR)-positive tumor cells, because of the high binding affinity of FA to the tumor-associated FR. We synthesized folate-mediated chondroitin sulfate (FA-PEG-ChS) for tumor cell targeting and non-folate-mediated naproxen-linked chondroitin sulfate (Nap-PEG-ChS) for comparison. Both the aforementioned polymers contain a PEG1000 spacer. We encapsulated an anticancer agent, DOX, during the formation of complexes with chitosan. Polyelectrolyte complexes (PEC) grafted with a fluorescent dye (FITC) served as a platform for online imaging cellular internalization. FR-positive KB and FR-deficient A549 cancer cells were tested. The concentration to kill 50% of the cells (IC₅₀) of DOX-loaded FA-complex was 1.53 μg/ml, in comparison to 0.91 μg/ml of free DOX. The overlaid fluorescent images of DOX and FITC on confocal laser scanning microscopy demonstrated the co-internalization of DOX and the complex nanoparticles into the cytoplasm of KB cells followed by a gradual release of DOX.     

Self-assembled pH-responsive MPEG-b-(PLA-co-PAE) block copolymer micelles for anticancer drug delivery

A series of amphiphilic pH-responsive poly (ethylene glycol) methyl ether-b-(poly lactic acid-co-poly (β-amino esters)) (MPEG-b-(PLA-co-PAE)) block copolymers with different PLA/PAE ratios were designed and synthesized via a Michael-type step polymerization. The molecular structures of the copolymers were confirmed with (1)H NMR and gel permeation chromatography (GPC). These amphiphilic copolymers were shown to self-assemble into core/shell micelles in aqueous solution at low concentrations, and their critical micelle concentrations (CMC) in water were 1.2-9.5 mg/L. The pH-responsive PAE segment was insoluble at pH 7.4, but it became positively charged and soluble via protonation of amino groups at pH lower than 6.5. The average particle size and zeta potential of micelles increased from 180 nm and 15 mV to 220 nm and 40 mV, respectively, when the pH decreased from 7.4 to 5.0. Doxorubicin (DOX) was loaded into the core of these micelles with a high drug loading of 18%. The in vitro DOX release from the micelles was significantly accelerated when solution pH decreased from 7.4 to 5.0. DOX release in the first 10 h appeared to follow Fickian diffusion mechanism. Toxicity test showed that the copolymers had low toxicity whereas the DOX-loaded micelles remained high cytotoxicity for HepG2 cells. The results indicate the pH-sensitive MPEG-b-(PLA-co-PAE) micelle may be a potential hydrophobic drug delivery carrier for cancer targeting therapy with sustained release.     

Enhanced effect of pH-sensitive mixed copolymer micelles for overcoming multidrug resistance of doxorubicin

P-glycoprotein (P-gp) mediated drug efflux has been recognized as a key factor contributing to the multidrug resistance (MDR) in tumor cells. To address this issue, a new pH-sensitive mixed copolymer micelles system composed of hyaluronic acid-g-poly(l-histidine) (HA-PHis) and d-α-tocopheryl polyethylene glycol 2000 (TPGS2k) copolymers was developed to co-deliver doxorubicin (DOX) and TPGS2k into drug-resistant breast cancer MCF-7 cells (MCF-7/ADR). The DOX-loaded HA-PHis/TPGS2k mixed micelles (HPHM/TPGS2k) were characterized to have a unimodal size distribution, high DOX loading content and a pH dependent drug release profile due to the protonation of poly(l-histidine). As compared to HA-PHis micelles (HPHM), the HPHM/TPGS2k showed higher and comparable cytotoxicity against MCF-7/ADR cells and MCF-7 cells, respectively. The enhanced MDR reversal effect was attributed to the higher amount of cellular uptake of HPHM/TPGS2k in MCF-7/ADR cells than HPHM, arising from the inhibition of P-gp mediated drug efflux by TPGS2k. The measurements of P-gp expression level and mitochondrial membrane potential indicate that the blank HPHM/TPGS2k inhibited P-gp activity by reducing mitochondrial membrane potential and depletion of ATP but without inhibition of P-gp expression. In vivo study of micelles in tumor-bearing mice indicate that HPHM/TPGS2k could reach the tumor site more effectively than HPHM. The pH-sensitive mixed micelles system has been demonstrated to be a promising approach for overcoming the MDR.     

Folate‐Conjugated Poly(N‐(2‐hydroxypropyl) methacrylamide)‐block‐Poly(benzyl methacrylate): Synthesis,Self‐Assembly,and Drug Release

Well‐defined amphiphilic diblock copolymers of poly(N‐(2‐hydroxypropyl)methacrylamide)‐block‐poly(benzyl methacrylate) (PHPMA‐b‐PBnMA) are synthesized using reversible addition–fragmentation chain transfer polymerization. The terminal dithiobenzoate groups are converted into carboxylic acids. The copolymers self‐assemble into micelles with a PBnMA core and PHPMA shell. Their mean size is <30 nm, and can be regulated by the length of the hydrophilic chain. The compatibility between the hydrophobic segment and the drug doxorubicin (DOX) affords more interaction of the cores with DOX. Fluorescence spectra are used to determine the critical micelle concentration of the folate‐conjugated amphiphilic block copolymer. Dynamic light scattering measurements reveal the stability of the micelles with or without DOX. Drug release experiments show that the DOX‐loaded micelles are stable under simulated circulation conditions and the DOX can be quickly released under acidic endosome pH.     

pH-sensitive micelles self-assembled from multi-arm star triblock co-polymers poly(ε-caprolactone)-b-poly(2-(diethylamino)ethyl methacrylate)-b-poly(poly(ethylene glycol) methyl ether methacrylate) for controlled anticancer drug delivery

A series of amphiphilic 4- and 6-armed star triblock co-polymers poly(ε-caprolactone)-b-poly(2-(diethylamino)ethyl methacrylate)-b-poly(poly(ethylene glycol) methyl ether methacrylate) (4/6AS-PCL-b-PDEAEMA-b-PPEGMA) were developed by a combination of ring opening polymerization and continuous activators regenerated by electron transfer atom transfer radical polymerization. The critical micelle concentration values of the star co-polymers in aqueous solution were extremely low (2.2–4.0 mg l^–1), depending on the architecture of the co-polymers. The self-assembled blank and doxorubicin (DOX)-loaded three layer micelles were spherical in shape with an average size of 60–220 nm determined by scanning electron microscopy and dynamic light scattering. The in vitro release behavior of DOX from the three layer micelles exhibited pH-dependent properties. The DOX release rate was significantly accelerated by decreasing the pH from 7.4 to 5.0, due to swelling of the micelles at lower pH values caused by the protonation of tertiary amine groups in DEAEMA in the middle layer of the micelles. The in vitro cytotoxicity of DOX-loaded micelles to HepG2 cells suggested that the 4/6AS-PCL-b-PDEAEMA-b-PPEGMA micelles could provide equivalent or even enhanced anticancer activity and bioavailability of DOX and thus a lower dosage is sufficient for the same therapeutic efficacy. The results demonstrate that the pH-sensitive multilayer micelles could have great potential application in delivering hydrophobic anticancer drugs for improved cancer therapy.     

Constructing doxorubicin-loaded polymeric micelles through amphiphilic graft polyphosphazenes containing ethyl tryptophan and PEG segments

By changing the molar ratio of hydrophilic and hydrophobic segments, a series of novel amphiphilic graft polyphosphazenes (PEG/EtTrp-PPPs) was synthesized via thermal ring-opening polymerization and a subsequent two-step substitution reaction of hydrophilic methoxyl polyethylene glycol (MPEG) and hydrophobic ethyl tryptophan (EtTrp). ¹H-Nuclear magnetic resonance and Fourier transform infrared studies validated the expected synthesis of copolymers. The copolymer composition was also confirmed by UV–visible spectrophotometry. The molar ratio of the segment PEG to group EtTrp was 1.33:0.67, 1.01:0.99 and 0.78:1.22, respectively. Micellization behavior of PEG/EtTrp-PPPs in an aqueous phase was characterized by fluorescence technique, dynamic light scattering and transmission electron microscopy. The critical micelle concentration (CMC) of the graft copolymer in aqueous solution was 0.158, 0.033 and 0.020 g l^?1, which decreased as the hydrophobic content in amphiphilic copolymers increased. Doxorubicin (DOX) was physically loaded into micelles prepared by an O/W emulsion method with a drug loading content increasing with DOX feeding. In vitro release of DOX from micelles can be accelerated in weak acidic solution. The results of cytotoxicity study using an MTT assay method with HeLa cell showed that amphiphilic graft polyphosphazenes were biocompatible while DOX-loaded micelles achieved comparable cytotoxicity with that of free DOX. In summary, these novel amphiphilic copolymers exhibited potential to be used as injectable drug carriers for tumor treatment.     

Self‐assembled UCST‐Type Micelles as Potential Drug Carriers for Cancer Therapeutics

A methoxy‐poly(ethylene glycol)‐block‐poly(acrylamide‐co‐acrylonitrile) (mPEG‐b‐P(AAm‐co‐AN)) amphiphilic copolymer exhibiting upper critical solution temperature (UCST) behavior is synthesized, and micelles from this copolymer are fabricated. It is found that the thermal responses of these micelles are tunable through balancing the hydrophobic/hydrophilic blocks in the copolymer. The size of the doxorubicin (DOX)‐loaded micelles is dependent on the hydrophobic interaction as well as hydrogen bonding between polymer and drug molecules. As a proof of concept, the drug release behavior is studied in vitro, and the cumulative release of DOX increases at temperature above the UCST of blank micelles. 3‐(4,5‐dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assays indicate that these polymers are non‐toxic towards human hepatic carcinoma cells (Bel 7402 cells) as well as human embryonic hepatocytes (L02 cells). DOX‐loaded micelles could effectively enter Bel 7402 cells in 2 h, and display much lower half inhibitory concentration compared with free DOX. These micelles may be exploited as a promising drug carrier for cancer therapeutics.

     

The tumor accumulation and therapeutic efficacy of doxorubicin carried in calcium phosphate-reinforced polymer nanoparticles

A mineral (calcium phosphate, CaP)-reinforced core-shell-corona micelle was evaluated as a nanocarrier of doxorubicin (DOX) for cancer therapy. The polymer micelles of poly(ethylene glycol)-b-poly(L-aspartic acid)-b-poly(L-phenylalanine) (PEG-PAsp-PPhe) in the aqueous phase provided the three distinct functional domains: the hydrated PEG outer corona for prolonged circulation, the anionic PAsp middle shell for CaP mineralization, and the hydrophobic PPhe inner core for DOX loading. CaP mineralization was performed by initial electrostatic localization of calcium ions at anionic PAsp shells, and the consequent addition of phosphate anions to trigger the growth of CaP. The mineralization did not affect the micelle size or the spherical morphology. The CaP-mineralized micelles exhibited enhanced serum stability. The DOX release from the DOX-loaded mineralized micelles (DOX-CaP-PM) at physiological pH was efficiently inhibited, whereas at an endosomal pH (pH 4.5), DOX release was facilitated due to the rapid dissolution of the CaP mineral layers in the middle shell domains. The in vivo tissue distribution and tumor accumulation of the DOX-CaP-PM that were labeled with a near-infrared fluorescent (NIRF) dye, Cy5.5, were monitored in MDA-MB231 tumor-bearing mice. Non-invasive real-time optical imaging results indicated that the DOX-CaP-PM exhibited enhanced tumor specificity due to the prolonged stable circulation in the blood and an enhanced permeation and retention (EPR) effect compared with the DOX-loaded nonmineralized polymer micelles (DOX-NPM). The DOX-CaP-PM exhibited enhanced therapeutic efficacy in tumor-bearing mice compared with free DOX and DOX-NPM. The CaP mineralization on assembled nanoparticles may serve as a useful guide for enhancing the antitumor therapeutic efficacy of various polymer micelles and nano-aggregates.     

Preparation of pH-sensitive zwitterionic nano micelles and drug controlled release for enhancing cellular uptake

Zwitterionic copolymers have exhibited high resistance to nonspecific protein adsorption and have wide applications in drug delivery systems. Herein, a pH-responsive poly(Lysine-alt-N,N′-bis(acryloyl) diaminohexane) was synthesized through the Michael addition polymerization between N, N′-bis(acryloyl) diaminohexane and lysine. Subsequently, nano micelles (NMs) were formed by self-assembly of the copolymer in an aqueous solution. The NMs showed a slightly negative charge in blood environment, but a positively charged surface in extracellular pH of tumor. This feature could be used to enhance permeability and retention effect, and reinforce tumor cell uptake. Vitro release studies revealed that the release of DOX from the DOX-loaded NMs was evidently faster at pH 5.0 than at pH 7.4. MTT assays revealed that NMs were nontoxic. Thus, these smart NMs were feasible candidates and could be potentially used in cancer chemotherapy.     

Multifunctional unimolecular micelles for cancer-targeted drug delivery and positron emission tomography imaging

A multifunctional unimolecular micelle made of a hyperbranched amphiphilic block copolymer was designed, synthesized, and characterized for cancer-targeted drug delivery and non-invasive positron emission tomography (PET) imaging in tumor-bearing mice. The hyperbranched amphiphilic block copolymer, Boltorn(?) H40-poly(L-glutamate-hydrazone-doxorubicin)-b-poly(ethylene glycol) (i.e., H40-P(LG-Hyd-DOX)-b-PEG), was conjugated with cyclo(Arg-Gly-Asp-D-Phe-Cys) peptides (cRGD, for integrin α(v)β(3) targeting) and macrocyclic chelators (1,4,7-triazacyclononane-N, N', N'-triacetic acid [NOTA], for (64)Cu-labeling and PET imaging) (i.e., H40-P(LG-Hyd-DOX)-b-PEG-OCH(3)/cRGD/NOTA, also referred to as H40-DOX-cRGD). The anti-cancer drug, doxorubicin (DOX) was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms (i.e., PLG) via a pH-labile hydrazone linkage to enable pH-controlled drug release. The unimolecular micelles exhibited a uniform size distribution and pH-sensitive drug release behavior. cRGD-conjugated unimolecular micelles (i.e., H40-DOX-cRGD) exhibited a much higher cellular uptake in U87MG human glioblastoma cells due to integrin α(v)β(3)-mediated endocytosis than non-targeted unimolecular micelles (i.e., H40-DOX), thereby leading to a significantly higher cytotoxicity. In U87MG tumor-bearing mice, H40-DOX-cRGD-(64)Cu also exhibited a much higher level of tumor accumulation than H40-DOX-(64)Cu, measured by non-invasive PET imaging and confirmed by biodistribution studies and ex vivo fluorescence imaging. We believe that unimolecular micelles formed by hyperbranched amphiphilic block copolymers that synergistically integrate passive and active tumor-targeting abilities with pH-controlled drug release and PET imaging capabilities provide the basis for future cancer theranostics.     

Tetraphenylsilane‐Cored Star‐Shaped Amphiphilic Block Copolymers for pH‐Responsive Anticancer Drug Delivery

Four‐arm star‐shaped poly[2‐(diethylamino)ethyl methacrylate]‐b‐poly[2‐hydroxyethyl methacrylate]s block copolymers using tetraphenylsilane (TPS) as a core with adjustable arm lengths are synthesized through two‐step atom transfer radical polymerizations. For comparison, a linear block copolymer with similar molecular weight is also prepared. The assembled star‐shaped copolymer micelles exhibit sizes of 102–139 nm and critical micelle concentrations of 1.49–3.93 mg L^?1. Moreover, the bulky and rigid TPS core is advantageous for propping up the four star‐shaped arms to generate large intersegmental space. As a result, the drug‐loading capacity in the micelles is up to 33.97 wt%, much surpassing the linear counterpart (8.92 wt%) and the previously reported star‐shaped copolymers prepared using pentaerythritol as the core. Furthermore, the micelles show sensitive pH‐responsive drug release when the pH changes from 7.4 to 5.0. The in vitro cytotoxicity to Hela cells indicates that the doxorubicin (DOX)‐loaded micelles have similar anticancer activity to the pristine DOX. The combination of excellent micelle stability, high drug‐loading, sensitive pH response, and good anticancer activity endows the copolymers with promising application in drug control delivery for anticancer therapy.