Biomaterials (v.32, #20)

Decreased material-activation of the complement system using low-energy plasma polymerized poly(vinyl pyrrolidone) coatings by Thomas E. Andersen; Yaseelan Palarasah; Mikkel-Ole Skjødt; Ryosuke Ogaki; Maike Benter; Mojagan Alei; Hans J. Kolmos; Claus Koch; Peter Kingshott (4481-4488).
In the current study we investigate the activation of blood complement on medical device silicone rubber and present a plasma polymerized vinyl pyrrolidone (ppVP) coating which strongly decreases surface-activation of the blood complement system. We show that uncoated silicone and polystyrene are both potent activators of the complement system, measured both as activated, deposited C3b and quantifying fluid-phase release of the cleavage fragment C3c. The ppVP coated silicone exhibits approximately 90% reduced complement activation compared to untreated silicone. Quartz crystal microbalance with dissipation (QCM-D) measurements show relatively strong adsorption of blood proteins including native C3 to the ppVP surface, indicating that reduction of complement activation on ppVP is neither a result of low protein adsorption nor lower direct C3-binding, and is therefore possibly a consequence of differences in the adsorbed protein layer composition. The alternative and classical complement pathways are barely detectable on ppVP while the lectin pathway through MBL/ficolin-2 deposition remains active on ppVP suggesting this pathway is responsible for the remaining subtle activation on the ppVP coated surface. The ppVP surface is furthermore characterized physically and chemically using scanning electron microscopy (SEM), x-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR), which indicates preservation of chemical functionality by the applied plasma process. Overall, the ppVP coating shows a potential for increasing complement–compatibility of blood-contacting devices.
Keywords: Silicone; Complement activation; Protein adsorption; Plasma polymerization; Vinyl pyrrolidone;

Neural lineage differentiation of embryonic stem cells within alginate microbeads by Lulu Li; Alexander E. Davidovich; Jennifer M. Schloss; Uday Chippada; Rene R. Schloss; Noshir A. Langrana; Martin L. Yarmush (4489-4497).
Cell replacement therapies, using renewable stem cell sources, hold tremendous potential to treat a wide range of degenerative diseases. Although many studies have established techniques to successfully differentiate stem cells into different mature cell lineages using growth factors or extracellular matrix protein supplementation in both two and three-dimensional configurations, they are often limited by lack of control and low yields of differentiated cells. Previously, we developed a scalable murine embryonic stem cell differentiation environment which maintained cell viability and supported ES cell differentiation to hepatocyte lineage cells. Differentiated hepatocyte function was contingent upon aggregate formation within the alginate microbeads. The present studies were designed to determine the feasibility of adapting the alginate encapsulation technique to neural lineage differentiation. The results of our studies indicate that by incorporating the soluble inducer, retinoic acid (RA), into the permeable microcapsule system, cell aggregation was decreased and neural lineage differentiation enhanced. In addition, we demonstrated that even in the absence of RA, differentiation could be directed away from the hepatocyte and toward the neural lineage by physical cell–cell aggregation blocking. In conjunction with the mechanical and physical characterization of the alginate crosslinking network, we determined that 2.2% alginate microencapsulation can be optimally adapted to ES neural differentiation. This study offers insights into targeting cellular differentiation toward both endodermal and ectodermal cell lineages, and could potentially be adaptable to differentiation of other stem cell types given the correct inducible factors and material properties.
Keywords: Alginate; Encapsulation; Embryonic stem cells; Microbead;

One-dimensional self-assembly of mouse embryonic stem cells using an array of hydrogel microstrands by Nurazhani Abdul Raof; Michael R. Padgen; Alison R. Gracias; Magnus Bergkvist; Yubing Xie (4498-4505).
The ability of embryonic stem (ES) cells to self-renew indefinitely and to differentiate into multiple cell lineages holds promise for advances in modeling disease progression, screening drugs and treating diseases. To realize these potentials, it is imperative to study self-assembly in an embryonic microenvironment, as this may increase our understanding of ES cell maintenance and differentiation. In this study, we synthesized an array of one-dimensional alginate gel microstrands and aqueous microstrands through an SU-8 filter device by means of capillary action. Furthermore, we investigated self-assembly behaviors and differentiation potentials of mouse ES cells cultured in microstrands of varying diameters. We found that microstrands with an aqueous interior facilitated high density cell culture and formed compact microtissue structures, while microstrands with gelled interiors promote smaller cell aggregate structures. In particular, we noticed that ES cells collected from one-dimensional aqueous microstrands favored the differentiation towards cell lineages of endoderm and mesoderm, whereas those from gelled microstrands preferred to differentiate into ectoderm and mesoderm lineages. In addition to providing a “liquid-like” tubular microenvironment to understand one-dimensional self-assembly process of ES cells, this alginate hydrogel microstrand system also offers an alternative way to manipulate the stem cell fate-decision using bioengineered microenvironments.
Keywords: Stem cell; Alginate; Hydrogel; Self-assembly; Microfiber; Microfluidics;

Biodegradable PDLLA/Chondroitin sulfate/Chitosan(PDLLA/CS/CHS) nerve conduits with potentially good biocompatibility and good mechanical property feasible for surgical manipulation have been developed in our previous work. The purpose of this study was to investigate their possible application in repairing damaged nerves and the effect of nerve growth factor (NGF). The PDLLA/CS/CHS/NGF nerve conduits were prepared by immobilizing NGF onto the PDLLA/CS/CHS nerve conduits with carbodiimide. Adult Sprague–Dawley (SD) rats weighing 200–250 g were used as the animal model. The conduits were employed to bridge the 10 mm defects in the sciatic nerve of the SD rats. Nerve conduction velocities (NCVs) were clearly detected in both nerve conduits after 3 months of implantation, indicating a rapid functional recovery for the disrupted nerves. The results of histological sections showed that the internal sides of the conduits were compact enough to prevent the connective tissues from ingrowth. Combined with the strong mechanical properties, good nerve regeneration ability and non-toxicity of its degradation products, PDLLA/CS/CHS nerve conduits would be expected to be useful materials to repair nerve damage and NGF can effectively promote the regeneration of peripheral nerve defect.
Keywords: PDLLA; Chondroitin sulfate; Chitosan; Peripheral nerve; Nerve conduits;

Permanent protection of PLG scaffold transplanted allogeneic islet grafts in diabetic mice treated with ECDI-fixed donor splenocyte infusions by Taba Kheradmand; Shusen Wang; Romie F. Gibly; Xiaomin Zhang; Samantha Holland; James Tasch; Jack G. Graham; Dixon B. Kaufman; Stephen D. Miller; Lonnie D. Shea; Xunrong Luo (4517-4524).
Allogeneic islet cell transplantation is a promising treatment for human type 1 diabetes. Currently, human islets are transplanted via intra-portal infusions. While successful, it leads to significant early islet attrition from instant blood-mediated inflammatory reaction. An extra-hepatic site was established by transplanting islet-loaded microporous poly(lactide-co-glycolide) (PLG) scaffolds into the epididymal fat pad in syngeneic islet transplant models. This study examined this technology in allogeneic islet transplantation and determined whether transplant tolerance could be effectively induced to protect PLG scaffold transplanted allogeneic islets. The efficacy of an established tolerance induction strategy using donor splenocytes treated with ethylcarbodiimide(ECDI) was tested. ECDI-fixed donor splenocytes were infused 7 days before and 1 day after islet transplantation. Immediate normoglycemia was restored, and treated mice maintained indefinite normoglycemia whereas untreated mice rejected islet grafts within 20 days of transplantation. Interestingly, efficacy of tolerance induction was superior in PLG scaffold compared with intra-portal transplanted islets. Protection of PLG scaffold islet allografts was associated with several mechanisms of immune regulation. In summary, PLG scaffolds can serve as an alternative delivery system for islet transplantation that does not impair tolerance induction. This approach of combining tolerance induction with scaffold islet transplantation has potential therapeutic implications for human islet transplantation.
Keywords: Islet; Transplantation; Scaffold; Poly(lactide-co-glycolide) (PLG); Allogeneic cell; Tolerance;

Human treated dentin matrix as a natural scaffold for complete human dentin tissue regeneration by Rui Li; Weihua Guo; Bo Yang; Lijuan Guo; Lei Sheng; Gang Chen; Ye Li; Qing Zou; Dan Xie; Xiaoxue An; Yali Chen; Weidong Tian (4525-4538).
An essential aspect of tooth tissue engineering is the identification of suitable scaffolding materials to support cell growth and tissue regeneration. Treated dentin matrix (TDM) from a rat has recently been shown to be a suitable scaffold for rat dentin regeneration. However, due to species-specific differences, it remains unclear whether a similar fabrication method can be extended to human TDM and human dentin regeneration. Therefore, this present study explored the biological response to a human TDM (hTDM) created using a modified dentin treatment method. Various biological characteristics, including cell proliferation, cell migration, cell viability, and cytotoxity were investigated. To assess the inductive capacity of hTDM, dental follicle cells (DFCs) were combined with hTDM and were implanted in vivo for 8 weeks in a mouse model. The resulting grafts were studied histologically. The results showed hTDM released dentinogenic factors, indicating that hTDM could play a sustained role in odontogenesis. DFC attachment, growth, viability, and cytotoxicity on the surface of hTDM showed a notable improvement over those on calcium phosphate controls. Most importantly, in vivo hTDM induced and supported regeneration of complete dentin tissues, which expressed dentin markers DSP and DMP-1. As cells in and around the regenerated dentin were positive for human mitochondria, implanted DFCs and hTDM were responsible for the regenerated dentin tissues. In conclusion, hTDM is indicated as an ideal biomaterial for human dentin regeneration.
Keywords: Dentin matrix; Stem cells; Microenvironment; Natural scaffold; Tooth; Regeneration;

Immobilization of soluble complement receptor 1 on islets by Nguyen M. Luan; Yuji Teramura; Hiroo Iwata (4539-4545).
Transplantation of pancreatic islets of Langerhans (islets) is a promising method to treat insulin-dependent diabetes mellitus. Control of complement activation is necessary to improve graft survival in alloislet and xenoislet transplantation. In this study, human soluble complement receptor 1 (sCR1) was immobilized on the islet cell surface through poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) without loss of islet cell viability or insulin secretion ability. sCR1 on islets effectively inhibits complement activation and protects islets against attack by xenoreactive antibodies and complement. This method will be an efficient means to control early islet loss in clinical islet transplantation and realize xenoislet transplantation in the future.
Keywords: Islet transplantation; Human soluble complement receptor 1 (sCR1); Surface modification; Poly(ethylene glycol)-conjugated phospholipid; Complement activation;

Long-term outcome of cryopreserved bone-derived osteoblasts for bone regeneration in vivo by Shaoyi Wang; Wenjie Zhang; Jun Zhao; Dongxia Ye; Chao Zhu; Yunhao Yang; Xiuli Zhang; Xiaojuan Sun; Chi Yang; Xinquan Jiang; Zhiyuan Zhang (4546-4555).
Cryopreserved bone-derived osteoblasts (CBOs) have been considered as a promising cell source for bone regeneration. Previous studies have demonstrated that CBOs had good proliferation and osteogenicity. However, the long-term outcome of CBOs in vivo still remains unknown. In this experiment, we applied CBOs combined with calcium phosphate cement (CPC) to augment maxillary sinus in canine, computer tomography, polychrome labeling, biomechanical tests, fluorescent immunohistochemistry staining and histological analysis were used to analyze the property and mineralization process of the tissue-engineered bone preclinical application. Our results showed that CBOs combined with CPC could promote bone regeneration, dramatically maintain the height, volume and biomechanical property of augmented maxillary sinus. Furthermore, the tissue-engineered bone was more mature than scaffold alone or autogenous bone, and bone formation and remodeling were still apparent 20 months postoperatively. Additionally, 4 months after surgery might be the suitable time point for implants placement in the regenerated bone. These results also indicate that cryopreserved bone may be a potential source of osteoblasts for maxillary sinus augmentation.
Keywords: Cryopreserved bone-derived osteoblasts; Maxillary sinus augmentation; Long-term; Bone regeneration;

In vivo efficacy of an intratumorally injected in situ-forming doxorubicin/poly(ethylene glycol)-b-polycaprolactone diblock copolymer by Yun Mi Kang; Gyeong Hae Kim; Jae Il Kim; Da Yeon Kim; Bit Na Lee; So Mi Yoon; Jae Ho Kim; Moon Suk Kim (4556-4564).
The effectiveness of systemically administered anticancer treatments is limited by difficulties in achieving therapeutic doses within tumors, a problem that is complicated by dose-limiting side effects to normal tissue. This work examined injectable in situ-forming gels as a localized drug-delivery system. An MPEG-PCL (MP) solution containing doxorubicin (Dox) existed in an emulsion-sol state at room temperature and rapidly gelled in vitro and in vivo at body temperature. The release of Dox from Dox-loaded MP gels was sustained in vitro over 20 days after an initial burst, indicating that the MP gel acted as a drug depot. Dox-loaded MP gels exhibited remarkable in vitro anti-proliferative activities against B16F10 cancer cells. In vivo experiments employing B16F10 cancer cell xenograft-bearing mice showed that a single intratumoral injection of Dox-loaded MP gel inhibited the growth of tumors as effectively as repeated injections of free Dox, and more effectively than a single dose of free Dox, or saline or gel alone. Consistent with the observed suppression of tumor growth, intratumorally injected free Dox or Dox released from Dox-loaded MP gels caused apoptosis of tumor cells. The tumor biodistribution of free Dox after 1 day was ∼90%, which dropped to ∼15% after 4 days. The biodistribution of Dox following a single injection of Dox-loaded MP gel was also ∼90% on day 1, but remained at ∼13%, even after 15 days. Only a small amount of Dox was found in other organ tissues following intratumoral injection, implying fewer off-target side effects.
Keywords: Injectable in situ gel; Tumors; Doxorubicin; Intratumoral injection;

The selective growth inhibition of oral cancer by iron core-gold shell nanoparticles through mitochondria-mediated autophagy by Ya-Na Wu; Li-Xing Yang; Xuan-Yu Shi; I-Chen Li; Joanna M. Biazik; Kyle R. Ratinac; Dong-Hwang Chen; Pall Thordarson; Dar-Bin Shieh; Filip Braet (4565-4573).
Nanoparticles with an iron core and gold shell (denoted “Fe@AuÓ”) have been reported to limit cancer-cell proliferation and therefore have been proposed as a potential anti-cancer agent. However, the underlying mechanisms are still unknown. In this study, we used flow cytometry, confocal fluorescence microscopy, and transmission electron microscopy to analyse the morphological and functional alterations of mitochondria in cancerous cells and healthy cells when treated with Fe@Au. It was found that Fe@Au caused an irreversible membrane-potential loss in the mitochondria of cancer cells, but only a transitory decrease in membrane potential in healthy control cells. Production of reactive oxygen species (ROS) was observed; however, additions of common ROS scavengers were unable to protect cancerous cells from the Fe@Au-induced cytotoxicity. Furthermore, iron elements, before oxidation, triggered mitochondria-mediated autophagy was shown to be the key factor responsible for the differential cytotoxicity observed between cancerous and healthy cells.
Keywords: Anti-cancer therapy; Autophagy; Cytotoxicity; Magnetic nanoparticles; Molecular biology; Reactive oxygen species;

Antitumor activity of liposomal ErbB2/HER2 epitope peptide-based vaccine constructs incorporating TLR agonists and mannose receptor targeting by Jean-Sébastien Thomann; Béatrice Heurtault; Steffen Weidner; Mélanie Brayé; Julien Beyrath; Sylvie Fournel; Francis Schuber; Benoît Frisch (4574-4583).
Synthetic and molecularly defined constructs containing the minimal components to mimic and amplify the physiological immune response are able to induce an efficient cytotoxic response. In the current study this approach was applied to the development of highly versatile liposomal constructs to co-deliver peptide epitopes in combination with TLR agonists in order to induce a specific anti-tumor cellular immune response against ErbB2 protein-expressing tumor cells. Liposomes containing ErbB2 p63–71 cytotoxic T lymphocyte (CTL) and HA307-319 T- helper (Th) peptide epitopes associated to innovative synthetic TLR2/1 (Pam3CAG) or TLR2/6 agonists (Pam2CAG and Pam2CGD), were injected in mice bearing ErbB2 protein-expressing tumor cells. Mannosylated ligands were also incorporated into the constructs to target antigen-presenting cells. We showed that the TLR2/6 agonists were more efficient than the TLR2/1 agonists for the eradication of tumors expressing ErbB2 protein. Furthermore, mannose-targeted liposomes displayed higher therapeutic efficiency against tumor allowing treatment with decreased quantities of both TLR ligands and peptide epitopes. Our results validated that antigen-associated mannosylated liposomes combined with efficient TLR ligands are effective vectors for vaccination against tumor. In this study we developed useful tools to evaluate the vaccination efficiency of various adjuvants and/or targeting molecules and their potential synergy.
Keywords: Liposome; Cancer vaccine; Peptide-based vaccine; Dendritic cell targeting; TLR ligands;

Targeted dual-contrast T 1- and T 2-weighted magnetic resonance imaging of tumors using multifunctional gadolinium-labeled superparamagnetic iron oxide nanoparticles by Hong Yang; Yeming Zhuang; Yun Sun; Antao Dai; Xiangyang Shi; Dongmei Wu; Fuyou Li; He Hu; Shiping Yang (4584-4593).
Development of a multifunctional nanoparticle (NP) system allowing for dual-contrast T 1- and T 2-weighted targeted magnetic resonance (MR) imaging of tumors could significantly improve the diagnosis accuracy. In this study, superparamagnetic silica-coated iron oxide core-shell nanoparticles (Fe3O4@SiO2 NPs) with a diameter of approximately 21 nm were synthesized via a thermal decomposition approach and were aminated through silanization. The amine-functionalized Fe3O4@SiO2 NPs enabled the covalent conjugation of a paramagnetic gadolinium complex (Gd-DTPA, DTPA: diethylenetriamine pentaacetic acid) and an arginine-glycine-aspartic acid (RGD) peptide as a targeting ligand onto their surface. The formed Fe3O4@SiO2(Gd-DTPA)-RGD NPs are water-dispersible, stable, and biocompatible as confirmed by MTT cell viability assay. Relaxivity measurements show that they have a T 1 relaxivity (r 1) of 4.2 mm −1 s−1 and T 2 relaxivity (r 2) of 17.4 mm −1 s−1 at the Gd/Fe molar ratio of 0.3:1, suggesting a possibility to use them as both T 1 positive and T 2 negative contrast agents. In vitro and in vivo MR imaging experiments show that the developed multifunctional Fe3O4@SiO2(Gd-DTPA)-RGD NPs enable targeted dual-contrast T 1- and T 2-weighted MR imaging of tumor cells over-expressing high-affinity αvβ 3 integrin in vitro and in vivo. Our results clearly indicate that the approach to forming multifunctional Fe3O4@SiO2(Gd-DTPA)-RGD NPs could be extended for fabricating other biologically active NPs for T 1- and T 2-weighted MR imaging of other biological systems with high accuracy.
Keywords: Dual-contrast; Targeted; Tumors; Magnetic resonance imaging; Gadolinium-labeled; Superparamagnetic iron oxide;

PEGylated cationic polymers have been extensively studied for substituting virus as gene delivery vehicles. These polymers can produce water-soluble polyionic complexes (polyplexes) with plasmid DNA (pDNA) and show enhanced stability compared to non-PEGylated polyplexes. However, PEGylation always diminishes the transfection efficiency of polyplexes probably due to poor cellular internalization of the particles and difficulty in releasing the pDNA cargo from the complexes intracellularly for gene expression. As non-ionic surfactants, Pluronic block copolymers have been shown to interact with plasma membrane and promote cellular uptake of various small molecules and biomacromolecules. To evaluate whether Pluronic could improve the transfection efficiency of polyplexes, Pluronic P85- and PEG-based cationomers comprising poly{N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide (P[Asp(DET)]) cationic blocks were synthesized and tested for their transfection ability. In this study, it was demonstrated that although the stability of the PEG-based polyplexes was better than that of the P85-based polyplexes based cationic polymers, the P85-based polyplex could achieve significantly higher transfection than the PEG counterparts. The improvement of gene delivering ability was shown to be correlated with the enhanced cellular internalization of the P85-based polyplexes.
Keywords: Pluronic; pDNA; Non-viral gene delivery; Polyplex;

Multifunctional and degradable nanogels encapsulating both model drug (fluorescently labeled dextran) and imaging reagent (monodisperse Fe3O4 nanoparticles) were developed by polymerizing zwitterionic monomers with a disulfide crosslinker. Results show that the nanogels have a hydrodynamic size of about 110 nm in saline solution and their size remained unchanged for over 6 months. After being conjugated with a targeting ligand, the nanogels showed a significant cellular uptake by human umbilical vein endothelial cells (HUVEC). The nanogels show low macrophage uptake, implying potential low interaction with the innate immune system. Upon entering the reducing intracellular environment, the disulfide bonds were efficiently cleaved, resulting in the spontaneous release of the encapsulated model drug and Fe3O4 nanoparticles. Magnetic resonance imaging (MRI) studies show that the encapsulation of multiple monodisperse Fe3O4 nanoparticles by the nanogels significantly enhanced their MRI performance (R2 relaxivity), while the disassembling of the Fe3O4 nanoparticles due to the nanogels degradation brings their R2 relaxivity back to that of their original monodisperse form. Furthermore, the degradation properties enable the removal of the disassembled nanogels from the body by renal clearance.
Keywords: Zwitterionic; Degradable; Reduction-sensitive; Renal clearance; Targeting; MRI (magnetic resonance imaging);

The mechanism of enhancement on oral absorption of paclitaxel by N-octyl-O-sulfate chitosan micelles by Ran Mo; Xiang Jin; Nan Li; Caoyun Ju; Minjie Sun; Can Zhang; Qineng Ping (4609-4620).
The overall objective of the present investigation was to demonstrate the effect of N-octyl-O-sulfate chitosan (NOSC) micelles on enhancing the oral absorption of paclitaxel (PTX) in vivo and in vitro, and identify the mechanism of this action of NOSC. In vivo, the oral bioavailability of PTX loaded in NOSC micelles (PTX-M) was 6-fold improved in comparison with that of an orally dosed Taxol®. In the Caco-2 uptake studies, NOSC micelles brought about a significantly higher amount of PTX accumulated in Caco-2 cells via both clathrin- and caveolae-mediated endocytosis, and NOSC had the effect on inhibiting PTX secreted by P-glycoprotein (P-gp), which was also proved by the studies on rhodamine 123 incorporated in NOSC micelles, fluorescence labeled micelles. The mechanism of NOSC on P-gp inhibition was demonstrated in connection with interfering the P-gp ATPase by NOSC rather than reducing the P-gp expression. Moreover, NOSC with the concentration approaching the critical micellar concentration (CMC) had the strongest effect on P-gp inhibition. In the Caco-2 transport studies, the presence of verapamil and NOSC both improved the transport of Taxol®, which further certified the effect of NOSC on P-gp inhibition, and PTX-M enhanced the permeability of PTX compared with Taxol®. The apparent permeability coefficient (Papp) of PTX-M decreased significantly at 4 °C in comparison with at 37 °C, which indicated a predominant active endocytic mechanism for the transport of PTX-M, a P-gp-independent way. Furthermore, the transcytosis of PTX-M was via clathrin-mediated rather than caveolae-mediated. In addition, the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers had no significant change during the transport study, which pointed out that NOSC had no effect on opening the intercellular tight junctions. Based on the obtained results, it is suggested that NOSC micelles might be a potentially applicable tool for enhancing the oral absorption of P-gp substrates.
Keywords: N-octyl-O-sulfate chitosan; Micelle; Paclitaxel; P-glycoprotein; Oral drug delivery;

Maltosylated polyethylenimine-based triple nanocomplexes of human papillomavirus 16L1 protein and DNA as a vaccine co-delivery system by Hee-Jeong Cho; Su-Eun Han; Saewon Im; Young Lee; Young Bong Kim; Taehoon Chun; Yu-Kyoung Oh (4621-4629).
To improve vaccine delivery, we herein designed a co-delivery system using a protein antigen and its encoding plasmid linked in nanocomplexes via maltosylated PEI (mPEI). Cationic mPEI was electrostatically complexed to a plasmid encoding the human papillomavirus (HPV) type 16L1 protein (pHPV16L1), and further complexed to a maltose binding protein (MBP)-fused human papillomavirus type 16L1 fusion protein (HPV16L1-MBP). The HPV16L1-MBP/mPEI/pHPV16L1 complexes were characterized by gel-retardation properties, zeta potentials and sizes. The intracellular co-delivery of protein and plasmid DNA vaccines was significantly higher for mPEI-based triple nanocomplexes than for a simple physical mixture of the proteins and DNA. Moreover, the cellular delivery of plasmid DNA using mPEI-based triple nanocomplexes resulted in higher expression levels comparable to those obtained using dual complexes of mPEI and the plasmid DNA. In vivo, co-immunization of mice with HPV16L1-MBP/mPEI/pHPV16L1 nanocomplexes triggered the highest levels of humoral immune responses among various vaccination groups. Moreover, the mPEI-based nanocomplexes significantly enhanced the number of interferon-γ producing CD8+ T cells compared with the use of mixed proteins and plasmid DNA. These results suggest that the effective cellular co-delivery of MBP-fused antigen proteins and plasmid DNA using maltosylated PEI-based triple nanocomplexes could enhance the immunogenicity of HPV16L1 vaccines.
Keywords: Vaccine co-delivery systems; Triple nanocomplexes; Maltosylated polyethylenimine; Maltose binding protein-fused protein; Human papillomavirus;

The structure–activity relationships between hydrophobic and hydrophilic modification on chitosan and resultant physicochemical properties along with performances in dealing with critical gene delivery barriers were investigated through amphiphilic linoleic acid(LA) and poly (β-malic acid) (PMLA) double grafted chitosan (LMC)/plasmid DNA (pDNA) nanocomplexes. LMC polymers with various LA and PMLA substitution degrees were synthesized and their hydrophilicity/hydrophobicity was characterized. Compared to chitosan, LMC nanoparticles retained the pDNA binding ability at pH 5.5 when they formed nanocomplexes with pDNA encoding enhanced green fluorescence protein (pEGFP) and the resultant complexes showed diameters below 300 nm. Hydrophobic LA and hydrophilic PMLA substitution contributed to suppressed non-specific adsorption, reduced interactions inside LMC/pDNA nanocomplexes, and enhanced pDNA dissociation. However, enzymatic degradation resistance, cell adsorption, and cellular uptake through clathrin-mediated pathway were promoted by hydrophobic LA grafting while being inhibited by hydrophilic PMLA substitution. In vitro transfection assay suggested the optimal LMC/pEGFP nanocomplexes mediated an 8.0-fold improved transfection compared to chitosan/pEGFP nanocomplexes. The 4.2-fold and 2.2-fold higher intramuscular gene expression in mice compared to chitosan/pEGFP and polyethyleneimine (PEI)/pEGFP nanocomplexes further demonstrated the superiority of LMC/pDNA nanocomplexes. Therefore, amphiphilic chitosan derivates with appropriate combination of hydrophobic and hydrophilic modification would be promising gene delivery nanocarriers.
Keywords: Amphiphilic chitosan based nanocarriers; Hydrophobic modification; Hydrophilic modification; Self-assembled nanocomplexes; Gene delivery; Transfection;

Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal release of the polyplexes by Marc Thibault; Mélina Astolfi; Nicolas Tran-Khanh; Marc Lavertu; Vincent Darras; Abderrazzak Merzouki; Michael D. Buschmann (4639-4646).
The optimal ratio of the polycation’s amine to DNA phosphate group (N:P) for efficient polymer-based transfection always employs excess polycation versus DNA. Most of the excess polycation remains free in solution, unassociated with the polyplexes, but is essential for efficient transfection. The mechanism by which excess polycation increases transfection efficiency is not identified. We hypothesised that excess chitosan facilitates intracellular lysosomal escape of the polyplexes. We highlight here the essential role of excess chitosan by rescuing poorly transfecting low N:P ratio polyplexes, by adding free chitosan before or after polyplex addition to cells. We examined polyplex uptake, the kinetics of rescue, intracellular trafficking, and the effects of lysosomotropic agents. We found the facilitating role of excess chitosan to be downstream of cellular uptake. Live-cell confocal quantification of intracellular trafficking revealed prolonged colocalisation of low N:P polyplexes within lysosomes, compared to shorter residence times for both rescued or N:P 5 samples, followed by observation of free pDNA in the cytosol. These data demonstrate that excess polycation mediates enhanced transfection efficiency by promoting the release of polyplexes from the endo-lysosomal vesicles, revealing a critical intracellular barrier overcome by excess polycation and suggesting possible avenues for further optimisation of polymer-based gene delivery.
Keywords: Chitosan; Gene transfer; DNA; Nanoparticle; Confocal microscopy;

Efficient gene delivery of primary human cells using peptide linked polyethylenimine polymer hybrid by Devaveena Dey; Mohammed Inayathullah; Andrew S. Lee; Melburne C. LeMieux; Xuexiang Zhang; Yi Wu; Divya Nag; Patricia Eliza De Almeida; Leng Han; Jayakumar Rajadas; Joseph C. Wu (4647-4658).
Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nanogels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly, when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of ‘reactive oxygen species’ (ROS) in the presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress.
Keywords: Polyethylenimine; DNA vectors; Primary human cells; Plasmids; Transfection agent; Stem cells;

The elucidation of gene transferring mechanism by ultrasound-responsive unmodified and mannose-modified lipoplexes by Keita Un; Shigeru Kawakami; Mitsuru Yoshida; Yuriko Higuchi; Ryo Suzuki; Kazuo Maruyama; Fumiyoshi Yamashita; Mitsuru Hashida (4659-4669).
The development of gene transfection methods enhancing the level of gene expression under simple and low-toxic condition is required for gene therapy in clinical. Our group has developed the ultrasound (US)-mediated gene transfection method using Man-PEG2000 bubble lipoplexes, which are US-responsive and mannose-modified gene carriers, and succeeded in obtaining the enhanced gene expression in mannose receptor-expressing cells selectively by the gene transfer using Man-PEG2000 bubble lipoplexes with US exposure in vitro and in vivo. Here, we investigated pDNA transferring mechanism followed by US exposure to unmodified and Man-PEG2000 bubble lipoplexes, in particular, focused on US exposure timing. Following investigation of intracellular transferring characteristics, a large amount of pDNA was transferred into the cytoplasm followed by US-mediated destruction of bubble lipoplexes in the gene transfer using both bubble lipoplexes with US exposure. Moreover, the effective gene expression was obtained without TNF-α production when US was exposed until 5 min after the addition of bubble lipoplexes. These findings suggest that the gene transfer using unmodified and Man-PEG2000 bubble lipoplexes with US exposure enables to transfer pDNA into the cytoplasm, and optimized US exposure timing is important to achieve the high level of gene expression and the low level of pro-inflammatory cytokine production.
Keywords: Bubble lipoplex; US exposure; Gene transfer; Endocytosis; Pro-inflammatory cytokine production;

The viscoelastic, hyperelastic and scale dependent behaviour of freshly excised individual skin layers by Michael L. Crichton; Bogdan C. Donose; Xianfeng Chen; Anthony P. Raphael; Han Huang; Mark A.F. Kendall (4670-4681).
Micro-devices using mechanical means to target skin for improved drug and vaccine delivery have great promise for improved clinical healthcare. Fully realizing this promise requires a greater understanding of key micro-biomechanical properties for each of the different skin layers – that are both the mechanical barriers and biological targets of these devices. Here, we performed atomic force microscopy indentation on a micro-nano scale to quantify separately, in fresh mouse skin, the viscous and elastic behaviour of the stratum corneum, viable epidermis and dermis. By accessing each layer directly, we examined the response to nanoindentation at sub-cellular and bulk-cellular scale. We found that the dermis showed greatest mechanical stiffness (elastic moduli of 7.33–13.48 MPa for 6.62 μm and 1.90 μm diameter spherical probes respectively). In comparison, the stratum corneum and viable epidermis were weaker at 0.75–1.62 MPa and 0.49–1.51 MPa respectively (again with the lower values resulting from indentations with the large probe 6.62 μm). The living cell layer of the epidermis (viable epidermis) showed greatest viscoelasticity – almost fully relaxing from shallow indentation – whilst the other layers reached a plateau after relaxing by around 40%. With small scale (sub-micron) AFM indentation, we directly determined the effects of different layer constituents – in particular, the dermis showed that some indents contacted collagen fibrils and others contacted ground substance/cellular areas. This work has far reaching implications for the design of micro-devices using mechanical means to deliver drugs or vaccines into the skin; providing key characterized mechanical property values for each constituent of the target delivery material.
Keywords: Skin; Epidermis; Dermis; AFM; Indentation; Mechanical properties;