Metastasis is the principal event leading to breast cancer death. Discovery of novel therapeutic approaches that are specific in targeting tumor metastasis factors while at the same time are an effective treatment of the tumor is urgently required. S100A4 protein is a key player in promoting metastasis and sequestrating the effect of tumor-suppressor protein p53. Here, a tumor microenvironment activated membrane fusogenic liposome was prepared to deliver rapidly anti-S100A4 antibody and doxorubicin into the cytoplasm directly in a fusion-dependent manner in order to bypass the cellular endocytosis to avoid the inefficient escape and degradation in the acidic endosome. After intracellular S100A4 blockage with anti-S100A4 antibody, the cytoskeleton of breast cancer 4T1 cells was rearranged and cell motility was suppressed. In the meantime, the antitumor effect of doxorubicin was enormously enhanced by reversing the effect of S100A4 on the sequestration of tumor-suppressor protein p53. Importantly, both local growth and metastasis of 4T1 cells were inhibited in a xenograft mouse model. Together, the speedy delivery of antibody and doxorubicin into cytoplasm based on a new membrane fusogenic liposome was an innovative approach for metastatic breast cancer treatment.Keywords: antibody; DOX; fusogenic liposome; metastasis; tumor microenvironment;

Multifunctional and multiresponsive hydrogels have presented a promising platform to design and fabricate smart devices for application in a wide variety of fields. However, their preparations often involve multistep preparation of multiresponsive polymer precursors, tedious reactions to introduce functional groups or sophisticated molecular designs. In this work, a multifunctional boronic acid-based cross-linker bis(phenylboronic acid carbamoyl) cystamine (BPBAC) was readily prepared from inexpensive commercially available 3-carboxylphenylboronic acid (CPBA) and cystamine dihydrochloride, which has the ability to cross-link the cis-diols and catechol-containing hydrophilic polymers to form hydrogels. Due to the presence of the reversible and dynamic boronate ester and disulfide bonds, the obtained hydrogels were demonstrated to not only possess pH, glucose, and redox triresponsive features, but also have autonomic self-healing properties under ambient conditions. Moreover, we can modulate the rheological and mechanical properties by simply adjusting the BPBAC amount. The features, such as commercially available starting materials, easy-to-implement approach, and versatility in controlling cross-linking network and mechanical properties, make the strategy described here a promising platform for fabricating multifunctional and smart hydrogels.

Long-term and daily subcutaneous injections of insulin for the treatment of insulin-dependent diabetic patients often lead to poor patient compliance and undesired complications. Phenylboronic acid (PBA)-based polymeric hydrogels have been widely considered as one of the most promising insulin delivery system to replace the frequent insulin injections. However, their applications are limited by clinically irrelevant glucose-responsive range, slow response rate, low tissue-adhesiveness and poor biodegradability, undesirable leakage at normoglycemic state. Herein, we report a novel implantable insulin hydrogel for glucose-regulated delivery of insulin based on a unique particle-hydrogel hybrid platform featuring fast glucose responsiveness at physiological pH, shear-thinning behavior for injection, tissue-adhesive function for long-lasting adherence, and full biodegradability for safe use. The system was thoroughly characterized both in vitro and in vivo and was demonstrated to hold these unique functions. Using streptozotocin-induced diabetic mice as a model, it was shown that a single subcutaneous injection of the insulin-loaded particle-hydrogel formulation led to quasi-steady-state blood glucose levels within the normal range for about two weeks. In addition, the preparation of the formulation only involved simple mixing and self-assembling processes, and thus it had great scalability and reproducibility for practical use. The highly feasible preparation, excellent performance, inherent biocompatibility and biodegradability make this novel composite hydrogel promising platform for diabetes therapy.Statement of SignificancePhenylboronic acid (PBA)-based polymeric hydrogels have been widely considered as one of the most promising insulin delivery system to replace the frequent insulin injections. However, these hydrogels, mostly based on a variety of PBA-containing acrylamide monomers, are still far from clinical reality. Building upon a unique particle-hydrogel hybrid platform, herein we report a novel implantable insulin storage and delivery system with multifunctionalities including fast glucose-sensitiveness at physiological pH, shear-thinning behavior for injection, tissue-adhesive function for long-lasting adherence, biodegradable materials for safe use and well-controlled insulin release. These unique functions were demonstrated through research both in vitro and in vivo. In addition, the preparation of the formulation was simple, and thus it had great scalability and reproducibility for practical use.Download high-res image (117KB)Download full-size image

Regioselectivity, a fundamental feature of radical addition reactions, has not received enough attention in reversible deactivation radical polymerization, especially for acrylates. However, in RAFT polymerization, the primary alkyl radicals, derived from “abnormal” head addition, will react with RAFT agents and produce non-active macro-RAFT agents, i.e. dead dormant species (DDS). Herein, a formula was proposed to correlate the content of DDS (α) with the incidence of head addition (ρ1) according to a detailed RAFT process involving head addition. And then, by combining a well-designed chain-extension reaction with gradient polymer elution chromatography (GPEC), the DDS in benzyl N-carbazolecarbodithioate-mediated RAFT polymerization of benzyl acrylate (BzA) were readily separated and quantitatively determined using the characteristic UV absorption of the N-carbazolecarbodithioate terminal. Based on these results, typically, the ρ1 of BzA at 60 °C was estimated to be 0.14%, demonstrating the utility of the method described to determine the incidence of head addition in radical polymerization. This study should be beneficial for a better understanding of RAFT processes, and meanwhile provides a powerful tool to determine regioselectivity of monomers in radical polymerization.

The preparations of covalently linked raspberry-like composite particles often suffer from uncontrolled particle shape and surface morphology, tedious reactions to introduce surface reactive groups, inefficient inter-particle reactions, and rigorous requirements for the formation of hierarchical structure. In this study, we developed a facile strategy to fabricate a kind of size-controlled, positively charged, alkoxysilanes-functionalized nanoparticles (Tsi-PDMAEMA-PSt NPs) via a combination of the ability of RAFT polymerization to design macromolecular architectures and the process of polymer self-assembly to produce well-defined NPs. Tsi-PDMAEMA-PSt NPs can effectively deposit on the outer surface of negatively charged silica microspheres and then form stable silica@polymer particles by the reaction between alkoxysilanes and surface silanols. The surface morphology, particle size, ζ-potential, structure stability as well as pH and thermo-responsiveness of the prepared composite particles were investigated. The results indicated that the prepared silica@polymer particles possessed unique raspberry-like surface structures with high stability and controllability. Moreover, the surface morphology and dispersion state of silica@polymer particles in water can respond to the change of pH and temperature. Consequently, considering the high simplicity and controllability, the design herein provided a promising route to prepare the long-stable raspberry-like composite microspheres with unique surface morphologies and stimuli-responsive properties for a wide range of possible applications.

To achieve higher therapeutic efficiency with catabatic side effects, desirable nanocarriers should be designed to retain the loaded drug tightly during the systemic circulation, but release the drug rapidly and efficiently upon endocytosis by tumor cells. Herein, to achieve “off-on” controlled delivery of DOX, novel amphiphilic polyelectrolyte/prodrug nanoparticles (NPs) with cooperative pH-sensitivity were constructed via synergistic electrostatic and hydrophobic interactions between slightly positively charged methoxy polyethylene glycol-b-(poly(2-(diisopropylamino) ethyl methacrylate-co-aminopropyl methacrylamide) (PEDPA) copolymer and negatively charged cis-aconityl-doxorubicin (CAD) prodrug (termed as PEDPA/CAD NPs). With polymer-prodrug synergistic noncovalent interactions, the drug loading content of PEDPA/CAD NPs could be improved up to 12.6% with favorable serum stability, and significantly lowered the drug leakage to 2.5% within 24 h at pH 7.4. However, nearly 80% of encapsulated drug could be released at pH 5.0 within 12 h, due to the cooperative effects of the protonation of PDPA blocks resulting in quick disassembly of NPs and the rapid hydrolysis of cis-aconityl linkage leading to charge-reverse of CAD. Moreover, the results of fluorescent microscopy imaging and flow cytometry measurements exhibited that DOX could be recovered and released rapidly from PEDPA/CAD NPs upon endocytosis and then exert therapeutic action in the cell nucleus. Importantly, the PEDPA/CAD NPs exhibited significantly higher antitumor efficiency in vivo with reduced nonspecific toxicity to normal tissues in comparation with free DOX. In summary, the NPs designed in this work, constructed by synergistic electrostatic and hydrophobic interactions with cooperative pH-sensitivity, which potentially resolved the dilemma between systemic stability and rapid intracellular drug release, would provide a promising nanomedicine platform for cancer therapy.Keywords: doxorubicin; nanoparticle; pH-sensitive; switchable delivery; tumor treatment

The effective preparation of photoresponsive polymers with precisely controlled location and number of photolabile units in the main chain is essential for their applications. In this study, a series of photocleavable well-defined triblock copolymers with the photocleavable middle block of poly(phenyl vinyl ketone) (PPVK) were readily synthesized by RAFT polymerization. The chain structure and chemical composition of copolymers were characterized by 1HNMR, FTIR and GPC. The well-controlled molecular weights and low polydispersity (<1.30) demonstrated the excellent controllability and living characteristics of the RAFT process for the polymerization of PVK. Then the photocleavage mechanism and kinetics of PPVK-functionalized copolymers were systematically investigated by tracking, fractionating and quantifying the photolysis products using gradient polymer elution chromatography (GPEC). The results not only confirmed the rapid photocleavability of PPVK-based polymers, but also firstly provided direct evidence for the proposed Norrish type reaction mechanism of the chain scission of PPVK. Moreover, the investigation of the effect of the PPVK chain on the photolysis kinetics demonstrated that the photodegradation rate of PPVK-based polymers can be controlled by adjusting the PPVK chain length in block copolymers. As a preliminary application study, the self-assembled micelles of the obtained PPVK-based amphiphilic polymers under light irradiation were found to undergo photo-triggered rapid disassembly and exhibited photo-controllable emulsifiability. In sum, the incorporation of the highly photolabile PPVK into block copolymers by RAFT polymerization provides a promising strategy for the construction of complex polymeric architectures or nanostructures with controllable photocleavability.

Polymeric nanoparticles assembled from the amphiphilic graft copolymer with cyclic benzylidene acetal-functionalized backbone and short poly(2-hydroxyethyl methacrylate) side chains can remain structurally stable at pH 7.4, but undergo stepwise disassembly in mildly acidic conditions due to the acid-triggered cleavage of the acetals, providing a promising nanocarrier for cancer therapy.

The application of PEG-b-PCL micelles was dampened by their inherent low drug-loading capability and relatively poor cell uptake efficiency. In this study, a series of novel PEG-b-PCL copolymers methoxy poly(ethylene glycol)-b-poly(ε-caprolactone-co-γ-dimethyl maleamidic acid -ε-caprolactone) (mPEG-b-P(CL-co-DCL)) bearing different amounts of acid-labile β-carboxylic amides on the polyester moiety were synthesized. The chain structure and chemical composition of copolymers were characterized by 1H NMR, Fourier transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC). mPEG-b-P(CL-co-DCL) with critical micellar concentrations (CMCs) of 3.2–6.3 μg/mL could self-assemble into stable micelles in water with diameters of 100 to 150 nm. Doxorubicin (DOX), a cationic hydrophobic drug, was successfully encapsulated into the polymer micelles, achieving a very high loading content due to electrostatic interaction. Then the stability, charge-conversional behavior, loading and release profiles, cellular uptake and in vitro cytotoxicity of free drug and drug-loaded micelles were evaluated. The β-carboxylic amides functionalized polymer micelles are negatively charged and stable in neutral solution but quickly become positively charged at pH 6.0, due to the hydrolysis of β-carboxylic amides in acidic conditions. The pH-triggered negative-to-positive charge reversal not only resulted in a very fast drug release in acidic conditions, but also effectively enhanced the cellular uptake by electrostatic absorptive endocytosis. The MTT assay demonstrated that mPEG-b-P(CL-co-DCL) micelles were biocompatible to HepG2 cells while DOX-loaded micelles showed significant cytotoxicity. In sum, the introduction of acid-labile β-carboxylic amides on the polyester block in mPEG-b-P(CL-co-DCL) exhibited great potentials for the modifications in the stability in blood circulation, drug solubilization, and release properties, as well as cell internalization and intracellular drug release.

To improve the poor compatibility among different components of Drug-in-adhesive type patch, two novel plasters (Drug-in-fiber and Drug-in-adhesive/fiber) were developed based on ibuprofen (IBU)-loaded fiber mats. These fibrous mats were fabricated via electrospinning of cellulose acetate/poly(vinylpyrrolidone) composites in a binary solvent of N,N-dimethyl acetamide/acetone. Physical status studies suggested that Drug-in-fiber could inhibit IBU re-crystallization, but the active ingredients were released at a relatively slow rate due to the dual-resistance of fiber mat and adhesive matrix. To overcome this shortcoming, Drug-in-adhesive/fiber was designed by coupling medicated hydrophilic pressure sensitive adhesive and IBU-loaded fiber mat. This method endowed Drug-in-adhesive/fiber a fast IBU release rate and high permeated drug amount though simulative skins. This design separated enhancer from adhesive matrix, which guaranteed Drug-in-adhesive/fiber excellent adhesion forces. Hence, the plasters based on medicated fiber mats improved the compatibility among patch components.

The pH-responsive micelles have enormous potential as nanosized drug carriers for cancer therapy due to their physicochemical changes in response to the tumor intracellular acidic microenvironment. Herein, a series of comb-like amphiphilic copolymers bearing acetal-functionalized backbone were developed based on poly[(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl) ethane methacrylate-co-poly(ethylene glycol) methyl ether methacrylate] [P(TTMA-co-mPEGMA)] as effective nanocarriers for intracellular curcumin (CUR) release. P(TTMA-co-mPEGMA) copolymers with different hydrophobic–hydrophilic ratios were prepared by one-step reversible addition fragmentation chain transfer (RAFT) copolymerization of TTMA and mPEGMA. Their molecular structures and chemical compositions were confirmed by 1H NMR, Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC). P(TTMA-co-mPEGMA) copolymers could self-assemble into nanosized micelles in aqueous solution and displayed low critical micelle concentration (CMC). All P(TTMA-co-mPEGMA) micelles displayed excellent drug loading capacity, due to the strong π–π conjugate action and hydrophobic interaction between the PTTMA and CUR. Moreover, the hydrophobic PTTMA chain could be selectively hydrolyzed into a hydrophilic backbone in the mildly acidic environment, leading to significant swelling and final disassembly of the micelles. These morphological changes of P(TTMA-co-mPEGMA) micelles with time at pH 5.0 were determined by DLS and TEM. The in vitro CUR release from the micelles exhibited a pH-dependent behavior. The release rate of CUR was significantly accelerated at mildly acidic pH of 4.0 and 5.0 compared to that at pH 7.4. Toxicity test revealed that the P(TTMA-co-mPEGMA) copolymers exhibited low cytotoxicity, whereas the CUR-loaded micelles maintained high cytotoxicity for HepG-2 and EC-109 cells. The results indicated that the novel P(TTMA-co-mPEGMA) micelles with low CMC, small and tunable sizes, high drug loading, pH-responsive drug release behavior, and good biocompatibility may have potential as hydrophobic drug delivery nanocarriers for cancer therapy with intelligent delivery.

In living polymerization, the inevitable dead chain impurities, due to bimolecular termination and side reactions, are not only an obstacle for the synthesis of pure block copolymers but also an impairment in the application performance of the resulting polymer materials. In this paper, a simple separation and quantification method based on gradient polymer elution chromatography (GPEC) was developed to experimentally investigate the dead species in RAFT polymerization of styrene. The RAFT-prepared polystyrene (PSt) was extended with methyl acrylate in a specially-designed chain extension reaction, in which the living PSt chains were extended and transformed into block copolymer, but the remaining dead PSt chains (D-PSt) will remain constant and can be separated and quantified by GPEC. The systematic experimental investigation of the effect of various polymerization parameters on the mass fraction of dead species (fD-PSt) in RAFT-prepared polystyrene (PSt) was implemented. The results clearly demonstrated the initiator concentration and RAFT agent concentration can exhibit a linear and inversely proportional dependence on fD-PSt, respectively. In addition, the dead chains were found to be formed throughout the entire RAFT process. These unambiguous experimental data confirmed the previous theoretical calculation and model prediction, which may be beneficial to understand RAFT processes, optimize polymerization conditions and to minimize dead polymer contaminations. Moreover, the method based on GPEC proposed here was able to fractionate and quantify the dead and living species, which can serve as a powerful tool in the mechanistic study of the living radical polymerizations.

A novel biodegradable amphiphilic diblock copolymer methoxy poly(ethylene glycol)-b-poly(ε-caprolactone-co-γ-hydroxyl-ε-caprolactone) (mPEG-b-P(CL-co-HCL)) bearing pendant hydroxyl groups on the PCL block was prepared. The hydroxyl groups were formed through the reduction of ketones by sodium borohydride without protection and deprotection. The obtained polymers were well characterized by 1H NMR, Fourier transform infrared (FT-IR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and contact angle measurement. mPEG-b-P(CL-co-HCL) could self-assemble into stable nanoparticles (NPs) with critical micellar concentrations (CMC) of 6.3 × 10–4 ∼ 8.1 × 10–4 mg/mL. The NPs prepared from mPEG-b-P(CL-co-HCL) were spherical in shape with diameters about 100 to 140 nm. The hydrophobic doxorubicin (DOX) was chosen as a drug model and successfully encapsulated into the NPs. The encapsulation efficiency and release kinetics of DOX were investigated. The results indicated that the introduction of hydroxyl groups onto the core-forming block could decrease the hydrophobicity of copolymers, thus improving the storage stability of NPs in aqueous solution. Moreover, higher loading capacity and slower in vitro release of DOX were observed, which was due to the hydrogen-bonding formation between DOX and hydroxyl groups. Meanwhile, the MTT assay demonstrated that the blank NPs were biocompatible to HepG2 cell,s while free DOX and DOX-loaded NPs showed significant cytotoxicity against the cells. Moreover, Compared to the free DOX, the DOX-loaded NPs were more efficiently internalized by HepG2 cells. In sum, the introduction of hydroxyl groups on the polyester block in mPEG-b-P(CL-co-HCL) exhibited great potentials for modifications in the stability, drug solubilization, and release properties of NPs.