This study aims to fabricate and deposit nanoscale multilayers on polyetheretherketone (PEEK) to improve cell adhesion and osseointegration. Bio-activated PEEK constructs were designed with prepared surface of different layers of polystyrene sulfonate (PSS) and polyallylamine hydrochloride (PAH) multilayers. Irregular morphology was found on the 5 and 10-layer PEEK surfaces, while “island-like” clusters were observed for 20-layer (20 L) multilayers. Besides, the 20 L PEEK showed more hydrophilic feature than native PEEK, and the surface contact angle reduced from 39.7° to 21.7° as layers increased from 5 to 20. In vitro, modified PEEK allowed excellent adhesion and proliferation of bone marrow stromal cells, and induced higher cell growth rate and alkaline phosphatase level. In vivo, this bio-active PEEK exhibited significantly enhanced integration with bone tissue in an osteoporosis rabbit model. This work highlights layer-by-layer self-assembly as a practical method to construct bio-active PEEK implants for enhanced osseointegration.Despite being currently used in spine and craniofacial surgeries, Polyetheretherketone (PEEK) is usually considered as a bio-inert material. Nanoscale PAH/PSS multilayers on the surface of PEEK, which were fabricated by LbL self-assembly, improved cell adhesion, viability, and proliferation in vitro. In vivo, enhanced osseointegration of 20-layer PEEK was observed in osteoporosis rabbit model. This work highlights layer-by-layer self-assembly as a practical alternative to construct bio-active PEEK implants for efficient osseointegration.Download high-res image (158KB)Download full-size image

Herein, comb-shaped polycations with neutral dextran as the main chain and folate-coupled bioreducible poly(urethane amine) (PUA) as the graft were designed and prepared as non-viral vectors for intravenous gene delivery targeting in tumor-bearing nude mice. Herein, primary amine-terminal PUAs with varied degrees of polymerization (DP) were prepared and then conjugated to dextrans with different molecular weights (5 kDa or 10 kDa), producing comb-shaped dextran-PUA polycations (denoted as Dex-PUA). The terminal group of the PUA graft could be further modified with folate, yielding folate-coupled Dex-PUA (denoted as Dex-PUA-FA). These comb-shaped polycations can condense genes into colloidal stable polyplexes under physiological conditions. However, these nano-polyplexes liberated genes in response to a reductive intracellular environment. In vitro transfection experiments showed that Dex10k-PUA40-FA, with 10 kDa dextran and a PUA oligomer with DP 40, induced the best transfection efficiency against SKOV-3 ovarian cancer cells in 10% serum. In vivo, the Dex10k-PUA40-FA polyplexes were applicable for intravenous gene delivery targeting SKOV-3 tumors in nude mice, affording a higher level of gene accumulation in the tumor as compared to Dex10k-PUA40 polyplexes lacking folate. Additionally, in vivo gene therapy showed that using a small hairpin RNA-silencing vascular endothelial growth factor, the Dex10k-PUA40-FA polyplexes exerted significant growth inhibition of SKOV-3 tumors with negligible systemic toxicity. The results of this study highlight dextranated PUA as a safe and robust gene vector for non-viral cancer gene therapy.

A group of poly(ethylene glycol)-poly(disulfide carbamate amine) (PEG–SSPCA) diblock copolymers is designed, prepared and successfully applied for intracellular dual-responsive drug delivery. PEG–SSPCA copolymers can be obtained by polycondensation reaction between 2,2′-dithiodiethanol bis(p-nitrophenyl carbonate) and a mixture of amino-terminal PEG (Mw = 5k) and tertiary amine-containing primary diamine. The copolymers self-assemble to form stable nanoscale micelles under physiological conditions and the micelles may undergo rapid destabilization under acidic or reductive conditions. The micelles based on the copolymer having a 1,4-bis(3-aminopropyl)piperazine (BAP) residue (termed as PEG–SSBAP) can carry anti-cancer drugs, doxorubicin (Dox) with the drug loading content of 5.7 ± 1%. The in vitro accumulative drug release test of Dox-loaded PEG–SSBAP micelles manifests slow drug release under physiological conditions and accelerated drug release in an acidic or reductive environment, but sufficient drug release in an acidic plus reductive environment. Confocal laser scanning microscopy imaging indicates that Dox-loaded PEG–SSBAP micelles are capable of delivering and liberating Dox into the cellular nucleus. In vitro, PEG–SSBAP micelles are of low toxicity against different cancer cells at a high concentration of 400 μg mL−1. However, Dox-loaded PEG–SSBAP micelles exert marked cytotoxicity against the cancer cells. In vivo, intravenous administration of the Dox-loaded micelles at a medium Dox dose of 2.5 mg kg−1 induces considerable growth inhibition of HepG2 tumor xenografted in nude mice with anti-cancer efficacy comparable to that of free Dox-chemotherapy but negligible systemic toxicity. The PEG–SSPCA block copolymer represents an efficient nano-carrier for controlled drug release and cancer therapy.

•Novel cationic gadolinium-chelated poly(urethane amide)s (GdCPUAs) are prepared.•GdCPUAs can induce a high transfection efficacy in different cancer cells.•GdCPUAs reveal good cyto-compatibility against cancer cells.•GdCPUAs may be applied as T1-contrast agents for magnetic resonance imaging.•GdCPUAs hold high potential for cancer theranostics.Theranostic nano-polyplexes containing gene and imaging agents hold a great promise for tumor diagnosis and therapy. In this work, we develop a group of new gadolinium (Gd)-chelated cationic poly(urethane amide)s for gene delivery and T1-weighted magnetic resonance (MR) imaging. Cationic poly(urethane amide)s (denoted as CPUAs) having multiple disulfide bonds, urethane and amide linkages were synthesized by stepwise polycondensation reaction between 1,4-bis(3-aminopropyl)piperazine and a mixture of di(4-nitrophenyl)-2, 2′-dithiodiethanocarbonate (DTDE-PNC) and diethylenetriaminepentaacetic acid (DTPA) dianhydride at varied molar ratios. Then, Gd-chelated CPUAs (denoted as GdCPUAs) were produced by chelating Gd(III) ions with DTPA residues of CPUAs. These GdCPUAs could condense gene into nanosized and positively-charged polyplexes in a physiological condition and, however, liberated gene in an intracellular reductive environment. In vitro transfection experiments revealed that the GdCPUA at a DTDE-PNC/DTPA residue molar ratio of 85/15 induced the highest transfection efficiency in different cancer cells. This efficiency was higher than that yielded with 25 kDa branched polyethylenimine as a positive control. GdCPUAs and their polyplexes exhibited low cytotoxicity when an optimal transfection activity was detected. Moreover, GdCPUAs may serve as contrast agents for T1-weighted magnetic resonance imaging. The results of this work indicate that biodegradable Gd-chelated cationic poly(urethane amide) copolymers have high potential for tumor theranostics.

The purpose of this work is to design a well-defined cationic dextran for intravenous gene delivery into tumor and examine the effect of the dextran on transfection efficacy in vivo. To this end, disulfide-linked dextran–linear polyethylenimine (Dex–SS–LPEI) conjugates were designed as non-viral vectors for intravenous gene delivery into tumor-bearing Balb/c nude mice. By coupling different molecular weights (2 kDa or 5 kDa) of LPEI disulfide pyridine to thiolated dextran (5 kDa or 10 kDa), Dex–SS–LPEI conjugates were prepared which have dextran as the main chain and disulfide-linked LPEI as the side chain. Dex–SS–LPEI conjugates can condense gene into nanosized polyplexes with moderate surface charge. Besides, the polyplexes of the conjugates have an improved colloidal stability under physiological conditions as compared to that of 22 kDa LPEI lacking dextran and can liberate gene by disulfide cleavage in an intracellular reducing environment. In vitro transfection experiments manifest that Dex–SS–LPEI conjugates mediate efficient gene transfection in different cells. The most efficient transfection in vitro was found for the conjugate with 5 kDa dextran and 5 kDa LPEI. Besides, Dex–SS–LPEI conjugates are practical for systemic gene delivery into tumor-bearing Balb/c nude mice by intravenous injection, affording comparable or higher transgene expression in HepG2 or SKOV-3 tumor than that yielded by 22 kDa LPEI as a positive control. Further, the polyplexes of the conjugate with 10 kDa dextran and 5 kDa LPEI can induce comparable transgene expression in the tumor but lower expression in the lung when compared to those of 22 kDa LPEI. The results of this study indicate that dextran plays a critical role in regulating in vivo gene delivery properties of Dex–SS–LPEI conjugates and transfection efficacy in tumor-xenografted nude mice. Dex–SS–LPEI conjugates have low cytotoxicity in vitro and cause no death of the mice, showing great potential as safe and highly efficient gene delivery vectors towards cancer gene therapy.

A folate-decorated, disulfide-based cationic dextran conjugate having dextran as the main chain and disulfide-linked 1,4-bis(3-aminopropyl)piperazine (BAP) residues as the grafts was designed and successfully prepared as a multifunctional gene delivery vector for targeted gene delivery to ovarian cancer SKOV-3 cells in vitro and in vivo. Initially, a new bioreducible cationic polyamide (denoted as pSSBAP) was prepared by polycondensation reaction of bis(p-nitrophenyl)-3,3′-dithiodipropanoate, a disulfide-containing monomer, and BAP. It was found that the pSSBAP was highly efficient for in vitro gene delivery against MCF-7 and SKOV-3 cell lines. Subsequently, two cationic dextran conjugates with different amounts of BAP residues (denoted as Dex-SSBAP6 and Dex-SSBAP30, respectively) were synthesized by coupling BAP to disulfide-linked carboxylated dextran or coupling pSSBAP-oligomer to p-nitrophenyl carbonated dextran. Both two conjugates were able to bind DNA to form nanosized polyplexes with an improved colloidal stability in physiological conditions. The polyplexes, however, were rapidly dissociated to liberate DNA in a reducing environment. In vitro transfection experiments revealed that the polyplexes of Dex-SSBAP30 efficiently transfected SKOV-3 cells, yielding transfection efficiency that is comparable to that of linear polyethylenimine or lipofectamine 2000. AlamarBlue assay showed that the conjugates had low cytotoxicity in vitro at a high concentration of 100 mg/L. Further, Dex-SSBAP30 has primary amine side groups and thus allows for folate (FA) conjugation, yielding FA-coupled Dex-SSBAP30 (Dex-SSBAP30-FA). It was found that Dex-SSBAP30-FA was efficient for targeted gene delivery to SKOV-3 tumor xenografted in a nude mouse model by intravenous injection, inducing a higher level of gene expression in the tumor as compared to Dex-SSBAP30 lacking FA and comparable gene expression to linear polyethylenimine as one of the most efficient polymeric vectors for intravenous gene delivery in vivo. Disulfide-based cationic dextran system thus has a high potential for intravenous gene delivery toward cancer gene therapy.Keywords: dextran; disulfide; ovarian cancer cells; systemic gene delivery; targeting;

In-situ forming hydrogels from thiolated glycol chitosan (GCH-SH) and vinyl sulfone-modified PEG (PL-VS) were designed, prepared and successfully applied as biodegradable, non-toxic bio-scaffolds for chondrocyte culture. The hydrogels could be formed in situ under physiological conditions via Michael-type addition between the GCH-SH and PL-VS at a low polymer concentration of 1–3% (w/v). Gelation times varied from 0.75 to 50 min, depending on the polymer concentration and the arm number of PEG-VS. Moreover, a high arm number and a high polymer concentration may lead to efficient network formation of GCH-SH/PEG-VS hydrogels. These hydrogels were found biodegradable in the presence of lysozyme, a cationic protein in the body, for a long period of time. Rheological studies indicated that these hydrogels generally displayed highly elastic property and had higher mechanical strength than those from thiolated hyaluronic acid/PEG-VS reported previously. SEM observation revealed that these hydrogels possessed well-interconnected microporous morphology. Besides these, the chondrocytes could be incorporated and homogeneously distributed in the hydrogel based on GCH-SH and 4-arm PL-VS. Importantly, after cell culture of 14 days, the chondrocytes in the hydrogel remained viable, as determined by a live–dead assay, and the cells kept their round chondrocytic phenotype. These results suggest that Michael-type addition is an effective method in the preparation of in-situ forming, biodegradable GCH-based hydrogels serving as bio-scaffolds for chondrocyte culture.Highlights► Thiolated glycol chitosan and vinyl sulfone-modified PEG were prepared successfully. ► Michael-type addition was applied to yield hydrogels based on glycol chitosan and PEG. ► These in-situ forming hydrogels are biodegradable, robust, highly elastic and porous. ► Chondrocytes encapsulated in the hydrogels maintain highly viable and round phenotype. ► Glycol chitosan-based hydrogels have high potential as scaffolds for cartilage repair.

A facile method for PEGylated bioreducible poly(amido amine)s is described by a one-pot Michael-type addition polymerization of N, N′-cystaminebisacrylamide (CBA) with a mixture of 4-amino-1-butanol (ABOL) and mono-tert-butoxycarbonyl (Boc) PEG diamine. By this approach, two Boc-amino-PEGylated p(CBA-ABOL) copolymers were obtained with the PEG/ABOL composition ratio of 1/10 (1a) and 1/6 (2a), respectively. These copolymers were characterized by 1H NMR and gel permeation chromatography. The PEGylated copolymers 1a, and its deprotected analog 1b with a terminal amino group at the PEG chain, were further evaluated as gene delivery vectors. The copolymers 1a and 1b condense DNA into nano-scaled PEGylated polyplexes (< 250 nm) with near neutral (2–5 mV, 1a) or slightly positive (9–13 mV, 1b) surface charge which remain stable in 150 mM buffer solution over 24 h. UnPEGylated polyplexes from p(CBA-ABOL), however, are relatively less stable and increase in size to more than 1 μm. The PEGylated polyplexes showed very low cytotoxicity in MCF-7 and NIH 3T3 cells and induced appreciable transfection efficiencies in the presence of 10% serum, although that are lower than those of p(CBA-ABOL) lacking PEG. The lower transfection efficiency of the PEGylated p(CBA-ABOL) polyplexes is discussed regarding the effect of PEGylation on endosomal escape of the PEGylated polyplexes.Research Highlights► PEGylated bioreducible poly(amido amine)s as gene carriers. ► PEGylated reducible poly(amido amine)s as safe and efficient gene carriers. ► One-pot synthesis of PEGylated reducible poly(amido amine)s as gene carriers. ► PEGylated reducible poly(amido amine)s for non-viral gene delivery. ► A facile preparation of PEGylated poly(amido amine)s as non-viral gene carriers.

•Injectable dextran hydrogels are prepared by metal-free click chemistry.•Azadibenzocyclooctyne-azide click chemistry is practical to prepare the hydrogel.•Click-crosslinked dextran hydrogels possess low cytotoxicity in chondrocytes.•Chondrocyte spheroids yield a high content of cartilage matrices in the hydrogel.•Injectable dextran hydrogels have high potential for cartilage tissue engineering.Injectable dextran-based hydrogels were prepared for the first time by bioorthogonal click chemistry for cartilage tissue engineering. Click-crosslinked injectable hydrogels based on cyto-compatible dextran (Mw = 10 kDa) were successfully fabricated under physiological conditions by metal-free alkyne-azide cycloaddition (click) reaction between azadibenzocyclooctyne-modified dextran (Dex-ADIBO) and azide-modified dextran (Dex-N3). Gelation time of these dextran hydrogels could be regulated in the range of approximately 1.1 to 10.2 min, depending on the polymer concentrations (5% or 10%) and ADIBO substitution degree (DS, 5 or 10) of Dex-ADIBO. Rheological analysis indicated that the dextran hydrogels were elastic and had storage moduli from 2.1 to 6.0 kPa with increasing DS of ADIBO from 5 to 10. The in vitro tests revealed that the dextran hydrogel crosslinked from Dex-ADIBO DS 10 and Dex-N3 DS 10 at a polymer concentration of 10% could support high viability of individual rabbit chondrocytes and the chondrocyte spheroids encapsulated in the hydrogel over 21 days. Individual chondrocytes and chondrocyte spheroids in the hydrogel could produce cartilage matrices such as collagen and glycosaminoglycans. However, the chondrocyte spheroids produced a higher content of matrices than individual chondrocytes. This study indicates that metal-free click chemistry is effective to produce injectable dextran hydrogels for cartilage tissue engineering.

The purpose of this work is to design a well-defined cationic dextran for intravenous gene delivery into tumor and examine the effect of the dextran on transfection efficacy in vivo. To this end, disulfide-linked dextran–linear polyethylenimine (Dex–SS–LPEI) conjugates were designed as non-viral vectors for intravenous gene delivery into tumor-bearing Balb/c nude mice. By coupling different molecular weights (2 kDa or 5 kDa) of LPEI disulfide pyridine to thiolated dextran (5 kDa or 10 kDa), Dex–SS–LPEI conjugates were prepared which have dextran as the main chain and disulfide-linked LPEI as the side chain. Dex–SS–LPEI conjugates can condense gene into nanosized polyplexes with moderate surface charge. Besides, the polyplexes of the conjugates have an improved colloidal stability under physiological conditions as compared to that of 22 kDa LPEI lacking dextran and can liberate gene by disulfide cleavage in an intracellular reducing environment. In vitro transfection experiments manifest that Dex–SS–LPEI conjugates mediate efficient gene transfection in different cells. The most efficient transfection in vitro was found for the conjugate with 5 kDa dextran and 5 kDa LPEI. Besides, Dex–SS–LPEI conjugates are practical for systemic gene delivery into tumor-bearing Balb/c nude mice by intravenous injection, affording comparable or higher transgene expression in HepG2 or SKOV-3 tumor than that yielded by 22 kDa LPEI as a positive control. Further, the polyplexes of the conjugate with 10 kDa dextran and 5 kDa LPEI can induce comparable transgene expression in the tumor but lower expression in the lung when compared to those of 22 kDa LPEI. The results of this study indicate that dextran plays a critical role in regulating in vivo gene delivery properties of Dex–SS–LPEI conjugates and transfection efficacy in tumor-xenografted nude mice. Dex–SS–LPEI conjugates have low cytotoxicity in vitro and cause no death of the mice, showing great potential as safe and highly efficient gene delivery vectors towards cancer gene therapy.

A group of poly(ethylene glycol)-poly(disulfide carbamate amine) (PEG–SSPCA) diblock copolymers is designed, prepared and successfully applied for intracellular dual-responsive drug delivery. PEG–SSPCA copolymers can be obtained by polycondensation reaction between 2,2′-dithiodiethanol bis(p-nitrophenyl carbonate) and a mixture of amino-terminal PEG (Mw = 5k) and tertiary amine-containing primary diamine. The copolymers self-assemble to form stable nanoscale micelles under physiological conditions and the micelles may undergo rapid destabilization under acidic or reductive conditions. The micelles based on the copolymer having a 1,4-bis(3-aminopropyl)piperazine (BAP) residue (termed as PEG–SSBAP) can carry anti-cancer drugs, doxorubicin (Dox) with the drug loading content of 5.7 ± 1%. The in vitro accumulative drug release test of Dox-loaded PEG–SSBAP micelles manifests slow drug release under physiological conditions and accelerated drug release in an acidic or reductive environment, but sufficient drug release in an acidic plus reductive environment. Confocal laser scanning microscopy imaging indicates that Dox-loaded PEG–SSBAP micelles are capable of delivering and liberating Dox into the cellular nucleus. In vitro, PEG–SSBAP micelles are of low toxicity against different cancer cells at a high concentration of 400 μg mL−1. However, Dox-loaded PEG–SSBAP micelles exert marked cytotoxicity against the cancer cells. In vivo, intravenous administration of the Dox-loaded micelles at a medium Dox dose of 2.5 mg kg−1 induces considerable growth inhibition of HepG2 tumor xenografted in nude mice with anti-cancer efficacy comparable to that of free Dox-chemotherapy but negligible systemic toxicity. The PEG–SSPCA block copolymer represents an efficient nano-carrier for controlled drug release and cancer therapy.

Herein, comb-shaped polycations with neutral dextran as the main chain and folate-coupled bioreducible poly(urethane amine) (PUA) as the graft were designed and prepared as non-viral vectors for intravenous gene delivery targeting in tumor-bearing nude mice. Herein, primary amine-terminal PUAs with varied degrees of polymerization (DP) were prepared and then conjugated to dextrans with different molecular weights (5 kDa or 10 kDa), producing comb-shaped dextran-PUA polycations (denoted as Dex-PUA). The terminal group of the PUA graft could be further modified with folate, yielding folate-coupled Dex-PUA (denoted as Dex-PUA-FA). These comb-shaped polycations can condense genes into colloidal stable polyplexes under physiological conditions. However, these nano-polyplexes liberated genes in response to a reductive intracellular environment. In vitro transfection experiments showed that Dex10k-PUA40-FA, with 10 kDa dextran and a PUA oligomer with DP 40, induced the best transfection efficiency against SKOV-3 ovarian cancer cells in 10% serum. In vivo, the Dex10k-PUA40-FA polyplexes were applicable for intravenous gene delivery targeting SKOV-3 tumors in nude mice, affording a higher level of gene accumulation in the tumor as compared to Dex10k-PUA40 polyplexes lacking folate. Additionally, in vivo gene therapy showed that using a small hairpin RNA-silencing vascular endothelial growth factor, the Dex10k-PUA40-FA polyplexes exerted significant growth inhibition of SKOV-3 tumors with negligible systemic toxicity. The results of this study highlight dextranated PUA as a safe and robust gene vector for non-viral cancer gene therapy.