Rapidly evolving protein technology has generated hundreds of therapeutic proteins that are promising for treating various human diseases. The clinical use of protein drugs remains, however, limited due to the absence of viable vehicles. Here, we report that anisamide-functionalized bioresponsive chimaeric nanopolymersomes (Anis-BCPs) can efficiently load granzyme B (GrB), a potent apoptotic protein, and enable targeted and efficacious protein therapy for H460 human lung cancer in vivo. Anis-BCPs are readily obtained from poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide-g-lipoic acid)-b-poly(acrylic acid) triblock copolymer. Notably, GrB-loaded Anis-BCPs a display superior antitumor effect toward sigma receptor-overexpressing H460 lung cancer cells (IC50 = 7.8 nM). The in vivo studies reveal that Anis-BCPs have a long circulation time and remarkable tumor accumulation. Interestingly, GrB-loaded Anis-BCPs at 6.24 nmol GrB equiv/kg dose, given either in four injections or one single injection, effectively inhibit H460 tumor growth and significantly improve the survival rate for mice. These robust, bioresponsive, and nontoxic chimaeric nanopolymersomes provide a potential platform for cancer protein therapy as well as basic research on intracellular functional proteins.

AbstractSmall interfering RNA (siRNA) offers a highly selective and effective pharmaceutical for various life-threatening diseases, including cancers. The clinical translation of siRNA is, however, challenged by its short plasma life, poor cell uptake, and cumbersome intracellular trafficking. Here, cNGQGEQc peptide-functionalized reversibly crosslinked chimaeric polymersomes (cNGQ/RCCPs) is shown to mediate high-efficiency targeted delivery of Polo-like kinase1 specific siRNA (siPLK1) to orthotopic human lung cancer in nude mice. Strikingly, siRNA is completely and tightly loaded into the aqueous lumen of the polymersomes at an unprecedentedly low N/P ratio of 0.45. cNGQ/RCCPs loaded with firefly luciferase specific siRNA (siGL3) or siPLK1 are efficiently taken up by α3β1-integrin-overexpressing A549 lung cancer cells and quickly release the payloads to the cytoplasm, inducing highly potent and sequence-specific gene silencing in vitro. The in vivo studies using nude mice bearing orthotopic A549 human lung tumors reveal that siPLK1-loaded cNGQ/RCCPs boost long circulation, superb tumor accumulation and selectivity, effective suppression of tumor growth, and significantly improved survival time. These virus-mimicking chimaeric polymersomes provide a robust and potent platform for targeted cancer siRNA therapy.

Cyclic RGD peptide-functionalized reversibly core-crosslinked biodegradable poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL) micelles (cRGD-RCCMs) were designed and developed for highly potent and targeted glioma chemotherapy. To achieve crosslinkable core, dithiolane-functionalized trimethylene carbonate (DTC) was incorporated into PCL block. Interestingly, cRGD-RCCMs displayed a high doxorubicin (DOX) loading content of ∼18 wt%, small hydrodynamic size of ∼50 nm, and excellent colloidal stability with minimum drug leakage under physiological conditions while fast DOX release under cytoplasmic-mimicking reductive environments. MTT, confocal microscopy and flow cytometry measurement results pointed out that cRGD-RCCMs with 30% cRGD surface density (cRGD30-RCCMs) showed an evident selectivity, efficient cytoplasmic drug release, and superior antitumor activity to clinically used pegylated liposomal doxorubicin (DOX-LPs) in αvβ3 integrin overexpressing U87MG glioblastoma cells. Strikingly, DOX-loaded cRGD30-RCCMs demonstrated a prolonged circulation time showing an elimination half-life of ∼4.7 h, three times exceeding that of the non-crosslinked counterparts, and a remarkably enhanced tumor accumulation of 7.7%ID/g. Furthermore, in vivo therapeutic studies revealed that DOX-loaded cRGD30-RCCMs effectively suppressed tumor growth, significantly prolonged survival time, and lessened side effects in subcutaneous U87MG glioblastoma-bearing nude mice. These reversibly core-crosslinked multifunctional biodegradable micelles might be developed into advanced and clinically viable targeted anticancer nanomedicines.Statement of SignificanceNanomedicines based on biodegradable micelles and nanoparticles offer a most promising treatment for malignant tumors. The therapeutic outcomes of current nanomedicines are, however, trimmed by their instability, low tumor retention, inefficient tumor cell uptake, and inferior drug release control. We report herein that cRGD-functionalized, rapidly glutathione-responsive, and reversibly core-crosslinked biodegradable micellar doxorubicin based on PEG-PCL block copolymer mediates potent and targeted glioma chemotherapy, affording significantly better treatment efficacy and lower systemic toxicity than the non-crosslinked micellar doxorubicin and clinically used pegylated liposomal doxorubicin controls. These reversibly core-crosslinked multifunctional biodegradable micelles have emerged as a robust, simple, versatile, and safe nanoplatform that might elegantly bridge the gap between the scientific and translational anticancer nanomedicine research.Download high-res image (131KB)Download full-size image

Liver cancer is a globally leading malignancy that has a poor five-year survival rate of less than 20%. The systemic chemotherapeutics are generally ineffective for liver cancers partly due to fast clearance and low tumor uptake. Here, we report that GE11 peptide functionalized polymersomal doxorubicin (GE11-PS-DOX) effectively targets and inhibits epidermal growth factor receptor (EGFR)-positive SMMC7721 orthotopic human liver tumor xenografts in mice. GE11-PS-DOX with a GE11 surface density of 10% displayed a high drug loading of 15.4 wt%, a small size of 78 nm, and glutathione-triggered release of DOX. MTT assays, flow cytometry and confocal microscopy studies revealed that GE11-PS-DOX mediated obviously more efficient DOX delivery into SMMC7721 cells than the non-targeting PS-DOX and clinically used liposomal doxorubicin (Lipo-DOX) controls. The in vivo studies showed that GE11-PS-DOX had a long circulation time and an extraordinary accumulation in the tumors (13.3 %ID/g). Interestingly, GE11-PS-DOX caused much better treatment of SMMC7721 orthotopic liver tumor-bearing mice as compared to PS-DOX and Lipo-DOX. The mice treated with GE11-PS-DOX (12 mg DOX equiv./kg) exhibited a significantly improved survival rate (median survival time: 130 days versus 70 and 38 days for PS-DOX at 12 mg DOX equiv./kg and Lipo-DOX at 6 mg DOX equiv./kg, respectively) and achieved 50% complete regression. Notably, GE11-PS-DOX induced obviously lower systemic toxicity than Lipo-DOX. EGFR-targeted multifunctional polymersomal doxorubicin with improved efficacy and safety has a high potential for treating human liver cancers.Statement of SignificanceLiver cancer is one of the top five leading causes of cancer death worldwide. The systemic chemotherapeutics and biotherapeutics generally have a low treatment efficacy for hepatocellular carcinoma partly due to fast clearance and/or low tumor uptake. Nanomedicines based on biodegradable micelle and polymersomes offer a most promising treatment for malignant liver cancers. Their clinical effectiveness remains, however, suboptimal owing to issues like inadequate systemic stability, low tumor accumulation and selectivity, and poor control over drug release. Here we report that GE11 peptide-functionalized, disulfide-crosslinked multifunctional polymersomal doxorubicin (GE11-PS-DOX) can effectively suppress the growth of orthotopic SMMC7721 human liver tumors in nude mice. They showed significantly decreased systemic toxicity and improved mouse survival rate with 3.4-fold longer median survival time as compared to clinically used pegylated liposomal doxorubicin (Lipo-DOX) and achieving 50% complete regression. GE11-PS-DOX, based on PEG-PTMC is biodegradable, nontoxic, and easy to prepare, appears as a safe, robust, versatile and all-function-in-one nanoplatform that has a high potential in targeted chemotherapy of EGFR expressed hepatocellular carcinoma.Download high-res image (84KB)Download full-size image

•DOX loaded HA-CM nanogels can be facilely prepared.•Both UV and NIR can trigger DOX release from HA-CM nanogels.•HA-CM nanogels enter CD44+ cancer cells via receptor mediated endocytosis.•NIR triggers nanogels to release DOX intracellularly causing tumor cell death.Hyaluronic acid (HA) is an endogenous polysaccharide that shows intrinsic targetability to CD44+ cancer cells. Here, we developed NIR and UV-responsive degradable nanogels from hyaluronic acid-g-7-N,N-diethylamino-4-hydroxymethylcoumarin (HA-CM) for CD44 targeted and remotely controlled intracellular doxorubicin (DOX) delivery. Nanometer-sized HA-CM nanogels could readily load DOX, and both NIR and UV irradiation could significantly enhance DOX release from the nanogels, resulting from light-triggered cleavage of urethane bonds that connect CM to HA. MTT assays showed that DOX-loaded HA-CM nanogels combined with NIR irradiation induced much higher antitumor activity to MCF-7 cells (CD44+) than to U-87MG cells (CD44-) and free HA pretreated MCF-7 cells. CLSM observations confirmed that DOX-loaded HA-CM nanogels were internalized by CD44+ cells via receptor mediated endocytosis mechanism, and intracellular DOX release was triggered by NIR. These HA-CM nanogels with easy preparation, CD44 targetability and photo-controlled intracellular drug release are interesting for cancer chemotherapy.Download high-res image (213KB)Download full-size image

Cancer nanomedicines are typically stealthed by a poly(ethylene glycol) layer that is important to obtain extended blood circulation and elevated tumor accumulation. PEG stealth, however, also leads to poor tumor cell selectivity and uptake thereby reducing treatment efficacy. Here, we report that biodegradable micelles with sheddable dendritic polyglycerol sulfate (dPGS) shells show an unusual tumor targetability and chemotherapy in vivo. The self-assembly of dPGS-SS-poly(ε-caprolactone) amphiphilic block copolymer with an Mn of 4.8–3.7 kg mol–1 affords negatively charged and small sized micelles (dPGS-SS-PCL Ms). dPGS-SS-PCL Ms reveal a low cytotoxicity, decent doxorubicin (DOX) loading, and accelerated drug release under a reductive condition. Notably, DOX-loaded dPGS-SS-PCL Ms exhibit a high tolerable dosage of more than 40 mg kg–1, a long plasma half-life of ca. 2.8 h, and an extraordinary tumor accumulation. Intriguingly, therapeutic results demonstrate that DOX-loaded dPGS-SS-PCL Ms induce complete tumor suppression, significantly improved survival rate, and diminishing adverse effects as compared to free drug (DOX·HCl) in MCF-7 human mammary carcinoma models. Dendritic polyglycerol sulfate with a superior tumor homing ability appears to be an attractive alternative to PEG in formulating targeted cancer nanomedicines.Keywords: biodegradable micelles; cancer chemotherapy; polyglycerol; reduction-sensitive; tumor targeting

Nanomedicines based on biodegradable micelles offer a most promising treatment for malignant tumors. Their clinical effectiveness, however, remains to be improved. Here, we report that self-crosslinkable and intracellularly decrosslinkable micellar nanoparticles (SCID-Ms) self-assembled from novel amphiphilic biodegradable poly(ethylene glycol)-b-poly(dithiolane trimethylene carbonate) block copolymer achieve high-efficiency targeted cancer chemotherapy in vivo. Interestingly, doxorubicin (DOX)-loaded SCID-Ms showed favorable features of superb stability, minimal drug leakage, long circulation time, triggered drug release inside the tumor cells, and an unprecedented maximum-tolerated dose (MTD) of over 100 mg DOX equiv./kg in mice, which was at least 10 times higher than free drug. The in vivo studies in malignant B16 melanoma-bearing C57BL/6 mice revealed that DOX-SCID-Ms at a dosage of 30 mg DOX equiv./kg could effectively suppress tumor growth and prolong mice survival time without causing obvious systemic toxicity. Moreover, DOX-SCID-Ms could be readily decorated with a targeting ligand like cRGD peptide. The biodistribution studies showed that cRGD20/DOX-SCID-Ms had a high tumor accumulation of 6.13% ID/g at 6 h post injection, which was ca. 3-fold higher than that for clinically used pegylated liposomal doxorubicin (DOX-LPs). Accordingly, cRGD20/DOX-SCID-Ms exhibited significantly better therapeutic efficacy and lower side effects than DOX-LPs in B16 melanoma-bearing mice. These self-regulating biodegradable micellar nanoparticles offer a robust, multifunctional and viable nanoplatform for targeted cancer chemotherapy.Cyclic RGD peptide-decorated disulfide-crosslinked micellar doxorubicin exhibits excellent stability, high maximum-tolerated dose, and superior targetability and therapeutic efficacy to pegylated liposomal doxorubicin in αvβ3 overexpressing B16 melanoma-bearing mice.

Pegylated liposomal doxorubicin (Lipo-Dox) is one of the few clinically used cancer nanomedicines. Here we show that tumor-homing, redox-responsive and reversibly crosslinked multifunctional biodegradable polymersomes are a better alternative to liposomes for Dox delivery. Cyclic peptide cNGQGEQc-decorated polymersomes (cNGQ-PS) are easily prepared with a small size and high Dox loading. Dox-loaded cNGQ-PS (cNGQ-PS-Dox) shows superb stability with minimal drug leakage under physiological conditions while spontaneous disassembly and quick drug release in response to 10 mM glutathione. MTT assays, flow cytometry and confocal microscopy clearly display efficient receptor-mediated internalization of cNGQ-PS-Dox, fast intracellular drug release, and high antitumor activity in α3β1 integrin-overexpressing A549 lung cancer cells. Intriguingly, cNGQ-PS-Dox presents a remarkably high maximum-tolerated dose of over 100 mg/kg, over 6-fold higher than Lipo-Dox. The in vivo pharmacokinetics and biodistribution studies reveal that cNGQ-PS-Dox has a long circulation time and significantly enhanced tumor accumulation (8.60%ID/g) as compared to Lipo-Dox and non-targeting PS-Dox controls. Notably, cNGQ-PS-Dox shows superior treatment of both subcutaneous and orthotopic A549 human lung cancer bearing nude mice to Lipo-Dox, resulting in effective tumor suppression, significantly improved survival time, and markedly reduced adverse effects. cNGQ-PS appears to be a clinically viable system for targeted lung cancer chemotherapy.

The therapeutic efficacy of nanoscale anticancer drug delivery systems is severely truncated by their low tumor-targetability and inefficient drug release at the target site. Here, we report the design and development of novel endosomal pH-activatable paclitaxel prodrug micelles based on hyaluronic acid-b-dendritic oligoglycerol (HA-dOG-PTX-PM) for active targeting and effective treatment of CD44-overexpressing human breast cancer xenografts in nude mice. HA-dOG-PTX-PM had a high drug content of 20.6 wt.% and an average diameter of 155 nm. The release of PTX was slow at pH 7.4 but greatly accelerated at endosomal pH. MTT assays, flow cytometry and confocal experiments showed that HA-dOG-PTX-PM possessed a high targetability and antitumor activity toward CD44 receptor overexpressing MCF-7 human breast cancer cells. The in vivo pharmacokinetics and biodistribution studies showed that HA-dOG-PTX-PM had a prolonged circulation time in the nude mice and a remarkably high accumulation in the MCF-7 tumor (6.19%ID/g at 12 h post injection). Interestingly, HA-dOG-PTX-PM could effectively treat mice bearing MCF-7 human breast tumor xenografts with little side effects, resulting in complete inhibition of tumor growth and a 100% survival rate over an experimental period of 55 days. These results indicate that hyaluronic acid-shelled acid-activatable PTX prodrug micelles have a great potential for targeted chemotherapy of CD44-positive cancers.

Novel reductively degradable α-amino acid-based poly(ester amide)-graft-galactose (SSPEA-Gal) copolymers were designed and developed to form smart nano-vehicles for active hepatoma-targeting doxorubicin (DOX) delivery. SSPEA-Gal copolymers were readily synthesized via solution polycondensation reaction of di-p-toluenesulfonic acid salts of bis-L-phenylalanine 2,2-thiodiethanol diester and bis-vinyl sulfone functionalized cysteine hexanediol diester with dinitrophenyl ester of adipic acid, followed by conjugating with thiol-functionalized galactose (Gal-SH) via the Michael addition reaction. SSPEA-Gal formed unimodal nanoparticles (PDI = 0.10 − 0.12) in water, in which average particle sizes decreased from 138 to 91 nm with increasing Gal contents from 31.6 wt% to 42.5 wt%. Notably, in vitro drug release studies showed that over 80% DOX was released from SSPEA-Gal nanoparticles within 12 h under an intracellular mimicking reductive conditions, while low DOX release (<20%) was observed for reduction-insensitive PEA-Gal nanoparticles under otherwise the same conditions and SSPEA-Gal nanoparticles under non-reductive conditions. Notably, SSPEA-Gal nanoparticles exhibited high specificity to asialoglycoprotein receptor (ASGP-R)-overexpressing HepG2 cells. MTT assays using HepG2 cells showed that DOX-loaded SSPEA-Gal had a low half maximal inhibitory concentration (IC50) of 1.37 μg mL−1, approaching that of free DOX. Flow cytometry and confocal laser scanning microscopy studies confirmed the efficient uptake of DOX-loaded SSPEA-Gal nanoparticles by HepG2 cells as well as fast intracellular DOX release. Importantly, SSPEA-Gal and PEA-Gal nanoparticles were non-cytotoxic to HepG2 and MCF-7 cells up to a tested concentration of 1.0 mg mL−1. These tumor-targeting and reduction-responsive degradable nanoparticles have appeared as an interesting multi-functional platform for advanced drug delivery.

In spite of their high potency and specificity, few protein drugs have advanced to the clinical settings due to lack of safe and efficient delivery vehicles. Here, novel anisamide-decorated pH-sensitive degradable chimaeric polymersomes (Anis-CPs) were designed, prepared, and investigated for efficient and targeted delivery of apoptotic protein, granzyme B (GrB), to lung cancer cells. Anis-CPs were readily prepared with varying Anis surface densities from anisamide end-capped poly(ethylene glycol)-b-poly(2,4,6- trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate)-b-poly(acrylic acid) (Anis-PEG-PTTMA-PAA) and PEG-PTTMA-PAA copolymers. Using cytochrome C (CC) as a model protein, Anis-CPs displayed high protein loading efficiencies (40.5–100%) and loading contents (up to 16.8 wt %). CC-loaded Anis-CPs had narrow distribution (PDI 0.04–0.13) and small sizes ranging from 152 to 171 nm, which increased with increasing CC contents. Notably, the release of proteins from Anis-CPs was accelerated under mildly acidic conditions, due to the hydrolysis of acetal bonds in PTTMA. MTT assays showed that GrB-loaded Anis-CPs (GrB-Anis-CPs) displayed high targetability to sigma receptor overexpressing cancer cells such as H460 and PC-3 cells. For example, GrB-Anis-CPs exhibited increasing antitumor efficacy to H460 cells with increasing Anis contents from 0 to 80%. The antitumor activity of GrB-Anis-CPs was significantly reduced upon pretreating H460 cells with haloperidol (a competitive antagonist). Notably, the half-maximal inhibitory concentrations (IC50) of GrB-Anis70-CPs were determined to be 6.25 and 5.94 nM for H460 and PC-3 cells, respectively, which were 2–3 orders of magnitude lower than that of chemotherapeutic drugs, such as paclitaxel. Flow cytometry studies demonstrated that GrB-Anis70-CPs induced widespread apoptosis of H460 cells. The confocal laser scanning microscopy (CLSM) experiments using FITC-labeled CC-loaded Anis-CPs confirmed fast internalization and intracellular protein release into H460 cells. GrB-Anis-CPs with high potency and specificity are particularly interesting for targeted therapy of lung cancers.

A novel and versatile family of enzymatically and reductively degradable α-amino acid-based poly(ester amide)s (SS-PEAs) were developed from solution polycondensation of disulfide-containing di-p-toluenesulfonic acid salts of bis-l-phenylalanine diesters (SS-Phe-2TsOH) with di-p-nitrophenyl adipate (NA) in N,N-dimethylformamide (DMF). SS-PEAs with Mn ranging from 16.6 to 23.6 kg/mol were obtained, depending on NA/SS-Phe-2TsOH molar ratios. The chemical structures of SS-PEAs were confirmed by 1H NMR and FTIR spectra. Thermal analyses showed that the obtained SS-PEAs were amorphous with a glass transition temperature (Tg) in the range of 35.2–39.5 °C. The in vitro degradation studies of SS-PEA films revealed that SS-PEAs underwent surface erosion in the presence of 0.1 mg/mL α-chymotrypsin and bulk degradation under a reductive environment containing 10 mM dithiothreitol (DTT). The preliminary cell culture studies displayed that SS-PEA films could well support adhesion and proliferation of L929 fibroblast cells, indicating that SS-PEAs have excellent cell compatibility. The nanoparticles prepared from SS-PEA with PVA as a surfactant had an average size of 167 nm in phosphate buffer (PB, 10 mM, pH 7.4). SS-PEA nanoparticles while stable under physiological environment undergo rapid disintegration under an enzymatic or reductive condition. The in vitro drug release studies showed that DOX release was accelerated in the presence of 0.1 mg/mL α-chymotrypsin or 10 mM DTT. Confocal microscopy observation displayed that SS-PEA nanoparticles effectively transported DOX into both drug-sensitive and -resistant MCF-7 cells. MTT assays revealed that DOX-loaded SS-PEA nanoparticles had a high antitumor activity approaching that of free DOX in drug-sensitive MCF-7 cells, while more than 10 times higher than free DOX in drug-resistant MCF-7/ADR cells. These enzymatically and reductively degradable α-amino acid-based poly(ester amide)s have provided an appealing platform for biomedical technology in particular controlled drug delivery applications.

cRGD-directed, NIR-responsive and robust AuNR/PEG–PCL hybrid nanoparticles (cRGD-HNs) were designed and developed for targeted chemotherapy of human glioma xenografts in mice. As expected, cRGD-HNs had excellent colloidal stability. The in vitro release studies showed that drug release from DOX-loaded cRGD-HNs (cRGD-HN-DOX) was minimal under physiological conditions but markedly accelerated upon NIR irradiation at a low power density of 0.2 W/cm2, due to photothermally induced phase transition of PCL regime. MTT assays showed that the antitumor activity of cRGD-HN-DOX in αvβ3 integrin over-expressed human glioblastoma U87MG cells was greatly boosted by mild NIR irradiation, which was significantly more potent than non-targeting HN-DOX counterpart under otherwise the same conditions and was comparable or superior to free DOX, supporting receptor-mediated endocytosis mechanism. The in vivo pharmacokinetics studies showed that cRGD-HN-DOX had much longer circulation time than free DOX. The in vivo imaging and biodistribution studies revealed that cRGD-HN-DOX could actively target human U87MG glioma xenograft in nude mice. The therapeutic studies in human U87MG glioma xenografts exhibited that cRGD-HN-DOX in combination with NIR irradiation completely inhibited tumor growth and possessed much lower side effects than free DOX. The Kaplan–Meier survival curves showed that all mice treated with cRGD-HN-DOX plus NIR irradiation survived over an experimental period of 48 days while control groups treated with PBS, cRGD-HN-DOX, cRGD-HNs with NIR irradiation, free DOX, or HN-DOX with NIR irradiation (non-targeting control) had short life spans of 15–40 days. Ligand-directed AuNR/PEG–PCL hybrid nanoparticles with evident tumor-targetability as well as superior spatiotemporal and rate control over drug release have emerged as an appealing platform for cancer chemotherapy in vivo.cRGD-functionalized and NIR-responsive AuNR/PEG–PCL hybrid nanoparticles mediate targeted delivery as well as remotely controlled release of doxorubicin into human glioblastoma xenografts in mice, leading to complete inhibition of tumor growth with little adverse effects and 100% mice survival over an experimental period of 48 days.

In this study, we designed and developed galactose-installed photo-crosslinked pH-sensitive degradable micelles (Gal-CLMs) for active targeting chemotherapy of hepatocellular carcinoma in mice. Gal-CLMs were readily obtained from co-self-assembly of poly(ethylene glycol)-b-poly(mono-2,4,6-trimethoxy benzylidene-pentaerythritol carbonate-co-acryloyl carbonate) (PEG-b-P(TMBPEC-co-AC)) and Gal-PEG-b-poly(ε-caprolactone) (Gal-PEG-b-PCL) copolymers followed by photo-crosslinking. Notably, paclitaxel (PTX)-loaded Gal-CLMs (Gal-PTX-CLMs) showed a narrow distribution (PDI = 0.08–0.12) with average sizes ranging from 92.1 to 136.3 nm depending on the Gal contents. The release of PTX from Gal-CLMs while inhibited at physiological pH was enhanced under endosomal pH conditions. MTT assays in asialoglycoprotein receptor (ASGP-R) over-expressing HepG2 cells demonstrated that half-maximal inhibitory concentration (IC50) values of Gal-PTX-CLMs decreased from 11.7 to 2.9 to 1.1 μg/mL with increasing Gal contents from 10% to 20% to 30%, supporting receptor-mediated endocytosis mechanism. The in vivo biodistribution studies in human hepatoma SMMC-7721 tumor-bearing nude mice displayed that Gal20-PTX-CLMs resulted in significantly enhanced drug accumulation in the tumors over non-targeting PTX-CLM counterpart. In accordance, Gal20-PTX-CLMs caused much greater tumor growth inhibition than non-targeting PTX-CLMs as well as non-crosslinking Gal20-PTX-NCLM controls (average tumor volume: ca. 35 mm3versus 144 mm3 and 130 mm3, respectively). Histological analysis showed that Gal20-PTX-CLMs induced more extensive apoptosis of tumor cells while less damage to normal liver and kidney compared to Taxol. Ligand-installed photo-crosslinked pH-responsive degradable micelles have a great potential for targeted cancer chemotherapy.Ligand-directed photo-crosslinked pH-sensitive degradable micelles simultaneously resolve dilemmas of stability versus intracellular drug release and stealth versus efficient internalization by target tumor cells, resulting in potent inhibition of tumor growth in vivo as well as alleviation of systemic side effects.

Redox and pH dual-responsive biodegradable micelles were developed based on poly(ethylene glycol)-SS-poly(2,4,6-trimethoxybenzylidene-pentaerythritol carbonate) (PEG-SS-PTMBPEC) copolymer and investigated for intracellular doxorubicin (DOX) release. PEG-SS-PTMBPEC copolymer with an Mn of 5.0–4.1 kg/mol formed micellar particles with an average diameter of 140 nm and a low polydispersity of 0.12. DOX was loaded into PEG-SS-PTMBPEC micelles with a decent drug loading content of 11.3 wt.%. The in vitro release studies showed that under physiological conditions only ca. 24.5% DOX was released from DOX-loaded micelles in 21 h. The release of DOX was significantly accelerated at pH 5.0 or in the presence of 10 mM glutathione (GSH) at pH 7.4, in which 62.8% and 74.3% of DOX was released, respectively, in 21 h. The drug release was further boosted under 10 mM GSH and pH 5.0 conditions, with 94.2% of DOX released in 10 h. Notably, DOX release was also facilitated by 2 or 4 h incubation at pH 5.0 and then at pH 7.4 with 10 mM GSH, which mimics the intracellular pathways of endocytosed micellar drugs. Confocal microscopy observation indicated that DOX was delivered and released into the nuclei of HeLa cells following 8 h incubation with DOX-loaded PEG-SS-PTMBPEC micelles, while DOX was mainly located in the cytoplasm for reduction-insensitive PEG-PTMBPEC controls. MTT assays revealed that DOX-loaded PEG-SS-PTMBPEC micelles had higher anti-tumor activity than reduction-insensitive controls, with low IC50 of 0.75 and 0.60 μg/mL for HeLa and RAW 264.7 cells, respectively, following 48 h incubation. PEG-SS-PTMBPEC micelles displayed low cytotoxicity up to a concentration of 1.0 mg/mL. These redox and pH dual-bioresponsive degradable micelles have appeared as a promising platform for targeted intracellular anticancer drug release.pH and redox dual-responsive biodegradable micelles trigger drug release not only in the acidic endosomal compartments but also in the reducing cytoplasms, resulting in superior antitumor effect.Figure optionsDownload full-size imageDownload high-quality image (294 K)Download as PowerPoint slide

Endosomal pH-activatable paclitaxel (PTX) prodrug micellar nanoparticles were designed and prepared by conjugating PTX onto water-soluble poly(ethylene glycol)-b-poly(acrylic acid) (PEG-PAA) block copolymers via an acid-labile acetal bond to the PAA block and investigated for potent growth inhibition of human cancer cells in vitro. PTX was readily conjugated to PEG-PAA with high drug contents of 21.6, 27.0, and 42.8 wt % (denoted as PTX prodrugs 1, 2, and 3, respectively) using ethyl glycol vinyl ether (EGVE) as a linker. The resulting PTX conjugates had defined molecular weights and self-assembled in phosphate buffer (PB, pH 7.4, 10 mM) into monodisperse micellar nanoparticles with average sizes of 158.3–180.3 nm depending on PTX contents. The in vitro release studies showed that drug release from PTX prodrug nanoparticles was highly pH-dependent, in which ca. 86.9%, 66.4% and 29.0% of PTX was released from PTX prodrug 3 at 37 °C in 48 h at pH 5.0, 6.0, and pH 7.4, respectively. MTT assays showed that these pH-sensitive PTX prodrug nanoparticles exhibited high antitumor effect to KB and HeLa cells (IC50 = 0.18 and 0.9 μg PTX equiv/mL, respectively) as well as PTX-resistant A549 cells. Notably, folate-decorated PTX prodrug micellar nanoparticles based on PTX prodrug 3 and 20 wt % folate-poly(ethylene glycol)-b-poly(d,l-lactide) (FA-PEG-PLA) displayed apparent targetability to folate receptor-overexpressing KB cells with IC50 over 12 times lower than nontargeting PTX prodrug 3 under otherwise the same conditions. Furthermore, PTX prodrug nanoparticles could also load doxorubicin (DOX) to simultaneously release PTX and DOX under mildly acidic pH. These acetal-linked PTX prodrug micellar nanoparticles have appeared as a highly versatile and potent platform for cancer therapy.

Hepatoma-targeting reduction-sensitive chimaeric biodegradable polymersomes were designed and developed based on galactose–poly(ethylene glycol)–poly(ε-caprolactone) (Gal-PEG-PCL), PEG–PCL–poly(2-(diethylamino)ethyl methacrylate) (PEG-PCL-PDEA, asymmetric), and PEG-SS-PCL for facile loading and triggered intracellular delivery of proteins. The chimaeric polymersomes formed from PEG-PCL-PDEA and PEG-SS-PCL had a monodisperse distribution with average sizes ranging from 95.5 to 199.2 nm depending on PEG-SS-PCL contents. Notably, these polymersomes displayed decent loading of bovine serum albumin (BSA), ovalbumin (OVA), and cytochrome C (CC) proteins likely due to presence of electrostatic and hydrogen bonding interactions between proteins and PDEA block located in the interior of polymersomes. The in vitro release studies showed that protein release was largely accelerated under a reductive condition containing 10 mM dithiothreitol (DTT). For example, ca. 77.2 and 22.1% of FITC-BSA were released from CP(SS50) (chimaeric polymersomes containing 50 wt % PEG-SS-PCL) at 37 °C in 12 h in the presence and absence of 10 mM DTT, respectively. Confocal microscopy showed that FITC-CC-loaded Gal-decorated CP(SS40) could efficiently deliver and release FITC-CC into HepG2 cells following 24 h treatment, in contrast to little or negligible fluorescence detected in HepG2 cells treated with FITC-CC-loaded nontargeting polymersomes or free CC. MTT assays revealed that CC-loaded Gal-decorated CP(SS40) exhibited apparent targetability and pronounced antitumor activity to HepG2 cells, in which cell viabilities decreased from 81.9, 60.6, 49.5, 42.2 to 31.5% with increasing Gal-PEG-PCL contents from 0, 10, 20, 30 to 40 wt %. Most remarkably, granzyme B-loaded Gal-decorated chimaeric polymersomes effectively caused apoptosis of HepG2 cells with a markedly low half-maximal inhibitory concentration (IC50) of 2.7 nM. These reduction-responsive chimaeric biodegradable polymersomes offer a multifunctional platform for efficient intracellular protein delivery.

The extracellular stability versus intracellular drug release dilemma has been a long challenge for micellar drug delivery systems. Here, core-crosslinked pH-sensitive degradable micelles were developed based on poly(ethylene glycol)-b-poly(mono-2,4,6-trimethoxy benzylidene-pentaerythritol carbonate-co-acryloyl carbonate) (PEG-b-P(TMBPEC-co-AC)) diblock copolymer that contains acid-labile acetal and photo-crossslinkable acryloyl groups in the hydrophobic polycarbonate block for intracellular paclitaxel (PTX) release. The micelles following photo-crosslinking while displaying high stability at pH 7.4 were prone to rapid hydrolysis at mildly acidic pHs of 4.0 and 5.0, with half lives of ca. 12.5 and 38.5 h, respectively. Notably, these micelles showed high drug loading efficiencies of 76.0–93.2% at theoretical PTX loading contents of 5–15 wt.%. Depending on drug loading contents, PTX-loaded micelles had average sizes varying from 132.2 to 171.6 nm, which were decreased by 17–22 nm upon photo-crosslinking. The in vitro release studies showed that PTX release at pH 7.4 was greatly inhibited by crosslinking of micelles. Notably, rapid drug release was obtained under mildly acidic conditions, in which 90.0% and 78.1% PTX was released in 23 h at pH 4.0 and 5.0, respectively. MTT assays showed that PTX-loaded crosslinked micelles retained high anti-tumor activity with a cell viability of 9.2% observed for RAW 264.7 cells following 72 h incubation, which was comparable to PTX-loaded non-crosslinked counterparts (cell viability 7.5%) under otherwise the same conditions, supporting efficient drug release from PTX-loaded crosslinked micelles inside the tumor cells. These core-crosslinked pH-responsive biodegradable micelles with superior extracellular stability and rapid intracellular drug release provide a novel platform for tumor-targeting drug delivery.Photo-crosslinked pH-sensitive degradable micelles while exhibit superior extracellular stability “actively” release paclitaxel under a mildly acidic condition mimicking that of the endo/lysosomal compartments, elegantly resolving extracellular stability and inrtracellular drug release dilemma of micellar drug delivery systems.

Reduction-responsive biodegradable micelles were developed from disulfide-linked dextran-b-poly(ε-caprolactone) diblock copolymer (Dex-SS-PCL) and applied for triggered release of doxorubicin (DOX) in vitro and inside cells. Dex-SS-PCL was readily synthesized by thiol-disulfide exchange reaction between dextran orthopyridyl disulfide (Dex-SS-py, 6000 Da) and mercapto PCL (PCL-SH, 3100 Da). Dynamic light scattering (DLS) measurements showed that Dex-SS-PCL yielded micelles with an average size of about 60 nm and a low polydispersity index (PDI 0.1−0.2) in PB (50 mM, pH 7.4). Interestingly, these micelles formed large aggregates rapidly in response to 10 mM dithiothreitol (DTT), most likely due to shedding of the dextran shells through reductive cleavage of the intermediate disulfide bonds. DOX could be efficiently loaded into the micelles with a drug loading efficiency of about 70%. Notably, the in vitro release studies revealed that Dex-SS-PCL micelles released DOX quantitatively in 10 h under a reductive environment, mimicking that of the intracellular compartments such as cytosol and the cell nucleus, whereas only about 27% DOX was released from reduction insensitive Dex-PCL micelles in 11.5 h under otherwise the same conditions and about 20% DOX released from Dex-SS-PCL micelles in 20 h under the nonreductive conditions. The cell experiments using fluorescence microscopy and confocal laser scanning microscopy (CLSM) showed clearly that DOX was rapidly released to the cytoplasm as well as to the cell nucleus. MTT studies revealed a markedly enhanced drug efficacy of DOX-loaded Dex-SS-PCL micelles as compared to DOX-loaded reduction-insensitive Dex-PCL micelles. These reduction-responsive biodegradable micelles have appeared highly promising for the targeted intracellular delivery of hydrophobic chemotherapeutics in cancer therapy.

Various functional biodegradable polymers were readily prepared based on novel cyclic carbonate monomers, acryloyl carbonate (AC) and methacryloyl carbonate (MAC), by combining ring-opening polymerization (ROP) and Michael-type conjugate addition. AC and MAC monomers were synthesized in four straightforward steps from 1,1,1-tris(hydroxymethyl)ethane with good overall yields (ca. 40%). AC and MAC were able to copolymerize with ε-caprolactone (ε-CL) and d,l-lactide (LA) in toluene at 110 °C using stannous octoate as a catalyst, yielding biodegradable copolymers with controlled (meth)acryloyl functional groups and molecular weights. The acryloyl groups were amenable to the Michael-type conjugate addition with varying thiol-containing molecules such as 2-mercaptoethanol, 3-mercaptopropanoic acid, cysteamine, cysteine, and arginine-glycine-aspartic acid-cysteine (RGDC) peptide under mild conditions, to provide biodegradable materials with vastly different functionalities (e.g., hydroxyl, carboxyl, amine, amino acid, and peptides) and properties (e.g., hydrophilicity, cell adhesion). Notably, 100% functionalization was achieved with 2-mercaptoethanol, cysteamine and cysteine. Initial cell culture studies demonstrated enhanced cell adhesion and growth on films containing functional RGDC peptides as compared to those of the parent copolymer. Therefore, combination of ROP and Michael-type conjugate addition provides a versatile access to diverse types of functional biodegradable materials.

Water-soluble temperature responsive triblock copolymers, poly(ethylene oxide)-b-poly(acrylic acid)-b-poly(N-isopropylacrylamide) (PEO-PAA-PNIPAM), were prepared in one pot by sequential reversible addition–fragmentation chain-transfer (RAFT) polymerization using a PEO–trithiocarbonate (PEO–S-1-dodecyl-S-(R,R-dimethyl-R-aceticacid) trithiocarbonate) as a macro chain transfer agent. The block copolymers with MnPEO of 5 kDa, MnPAA of 0.35–1.45 kDa, and MnPNIPAM varying from 11–39 kDa were freely soluble in water as unimers at room temperature, but quickly self-assembled into nano-sized vesicles (about 220 nm) when raising the solution temperature to 37 °C. The vesicular structure was confirmed by confocal scanning laser microscope (CSLM) and static light scattering (SLS) measurements. The size and size distribution of the polymersomes depended on the solution concentration, the molecular weight of PNIPAM, the equilibrium time and shaking. Interestingly, thus-formed vesicles could be readily cross-linked at the interface using cystamineviacarbodiimide chemistry. The crosslinked polymersomes, while showing remarkable stability against dilution, organic solvent, high salt conditions and change of temperature in water, were otherwise rapidly dissociated under reductive conditions mimicking intracellular environment. Notably, FITC–dextran, used as a model protein, was shown to be encapsulated into the polymersomes with an unprecedently high loading efficiency (>85 wt%). The release studies showed that most FITC–dextran was retained within the polymersomes after lowering the temperature to 25 °C. However, in the presence of 10 mM dithiothreitol (DTT), fast release of FITC–dextran was achieved. These reversibly crosslinked temperature responsive nano-sized polymersomes are highly promising as smart carriers for triggered intracellular delivery of biopharmaceutics such as pDNA, siRNA, pharmaceutical proteins and peptides.

Novel reductively degradable α-amino acid-based poly(ester amide)-graft-galactose (SSPEA-Gal) copolymers were designed and developed to form smart nano-vehicles for active hepatoma-targeting doxorubicin (DOX) delivery. SSPEA-Gal copolymers were readily synthesized via solution polycondensation reaction of di-p-toluenesulfonic acid salts of bis-L-phenylalanine 2,2-thiodiethanol diester and bis-vinyl sulfone functionalized cysteine hexanediol diester with dinitrophenyl ester of adipic acid, followed by conjugating with thiol-functionalized galactose (Gal-SH) via the Michael addition reaction. SSPEA-Gal formed unimodal nanoparticles (PDI = 0.10 − 0.12) in water, in which average particle sizes decreased from 138 to 91 nm with increasing Gal contents from 31.6 wt% to 42.5 wt%. Notably, in vitro drug release studies showed that over 80% DOX was released from SSPEA-Gal nanoparticles within 12 h under an intracellular mimicking reductive conditions, while low DOX release (<20%) was observed for reduction-insensitive PEA-Gal nanoparticles under otherwise the same conditions and SSPEA-Gal nanoparticles under non-reductive conditions. Notably, SSPEA-Gal nanoparticles exhibited high specificity to asialoglycoprotein receptor (ASGP-R)-overexpressing HepG2 cells. MTT assays using HepG2 cells showed that DOX-loaded SSPEA-Gal had a low half maximal inhibitory concentration (IC50) of 1.37 μg mL−1, approaching that of free DOX. Flow cytometry and confocal laser scanning microscopy studies confirmed the efficient uptake of DOX-loaded SSPEA-Gal nanoparticles by HepG2 cells as well as fast intracellular DOX release. Importantly, SSPEA-Gal and PEA-Gal nanoparticles were non-cytotoxic to HepG2 and MCF-7 cells up to a tested concentration of 1.0 mg mL−1. These tumor-targeting and reduction-responsive degradable nanoparticles have appeared as an interesting multi-functional platform for advanced drug delivery.

Water-soluble temperature responsive triblock copolymers, poly(ethylene oxide)-b-poly(acrylic acid)-b-poly(N-isopropylacrylamide) (PEO-PAA-PNIPAM), were prepared in one pot by sequential reversible addition–fragmentation chain-transfer (RAFT) polymerization using a PEO–trithiocarbonate (PEO–S-1-dodecyl-S-(R,R-dimethyl-R-aceticacid) trithiocarbonate) as a macro chain transfer agent. The block copolymers with MnPEO of 5 kDa, MnPAA of 0.35–1.45 kDa, and MnPNIPAM varying from 11–39 kDa were freely soluble in water as unimers at room temperature, but quickly self-assembled into nano-sized vesicles (about 220 nm) when raising the solution temperature to 37 °C. The vesicular structure was confirmed by confocal scanning laser microscope (CSLM) and static light scattering (SLS) measurements. The size and size distribution of the polymersomes depended on the solution concentration, the molecular weight of PNIPAM, the equilibrium time and shaking. Interestingly, thus-formed vesicles could be readily cross-linked at the interface using cystamineviacarbodiimide chemistry. The crosslinked polymersomes, while showing remarkable stability against dilution, organic solvent, high salt conditions and change of temperature in water, were otherwise rapidly dissociated under reductive conditions mimicking intracellular environment. Notably, FITC–dextran, used as a model protein, was shown to be encapsulated into the polymersomes with an unprecedently high loading efficiency (>85 wt%). The release studies showed that most FITC–dextran was retained within the polymersomes after lowering the temperature to 25 °C. However, in the presence of 10 mM dithiothreitol (DTT), fast release of FITC–dextran was achieved. These reversibly crosslinked temperature responsive nano-sized polymersomes are highly promising as smart carriers for triggered intracellular delivery of biopharmaceutics such as pDNA, siRNA, pharmaceutical proteins and peptides.