Biomaterials (v.32, #12)

A simplified genetic design for mammalian enamel by Malcolm L. Snead; Dan-Hong Zhu; Yaping Lei; Wen Luo; Pablo O. Bringas; Henry M. Sucov; Richard J. Rauth; Michael L. Paine; Shane N. White (3151-3157).
A biomimetic replacement for tooth enamel is urgently needed because dental caries is the most prevalent infectious disease to affect man. Here, design specifications for an enamel replacement material inspired by Nature are deployed for testing in an animal model. Using genetic engineering we created a simplified enamel protein matrix precursor where only one, rather than dozens of amelogenin isoforms, contributed to enamel formation. Enamel function and architecture were unaltered, but the balance between the competing materials properties of hardness and toughness was modulated. While the other amelogenin isoforms make a modest contribution to optimal biomechanical design, the enamel made with only one amelogenin isoform served as a functional substitute. Where enamel has been lost to caries or trauma a suitable biomimetic replacement material could be fabricated using only one amelogenin isoform, thereby simplifying the protein matrix parameters by one order of magnitude.
Keywords: Biomimetic material; Biomineralization; Fracture toughness; Genetic engineering; Mechanical properties; Molecular biology;

A perfluoropolyether corneal inlay for the correction of refractive error by Margaret D.M. Evans; Ruby K. Prakasam; Pravin K. Vaddavalli; Timothy C. Hughes; Warren Knower; John S. Wilkie; Keith M. McLean; Graham Johnson; Gail A. McFarland; Ruo Zhong Xie; Deborah F. Sweeney (3158-3165).
This study assessed the long-term biological response of a perfluoropolyether-based polymer developed as a corneal inlay to correct refractive error. The polymer formulation met chemical and physical specifications and was non-cytotoxic when tested using standard in vitro techniques. It was cast into small microporous membranes that were implanted as inlays into corneas of rabbits (n = 5) and unsighted humans (n = 5 + 1 surgical control) which were monitored for up to 23 and 48 months respectively. Overall, the inlays were well tolerated during study period with the corneas remaining clear and holding a normal tear film and with no increased vascularisation or redness recorded. Inlays in three human corneas continued past 48 months without sequelae. Inlays in two human corneas were removed early due to small, focal erosions developing 5 and 24 months post-implantation. Polymer inlays maintained their integrity and corneal position for the study duration although the optical clarity of the inlays reduced slowly with time. Inlays induced corneal curvature changes in human subjects that showed stability with time and the refractive effect was reversed when the inlay was removed. Outcomes showed the potential of a perfluoropolyether inlay as a biologically acceptable corneal implant with which to provide stable correction of refractive error.
Keywords: Ophthalmology; Cornea; Biocompatibility; In vivo test; Fluoropolymer;

Osteoconductive properties of poly(96L/4D-lactide)/beta-tricalcium phosphate in long term animal model by Guy Daculsi; Eric Goyenvalle; Ronan Cognet; Eric Aguado; Esa O. Suokas (3166-3177).
The objective of this study was to determine the effect of calcium phosphate mineral content on the bone in-growth at the expense of composite of co-polylactide polymer charged with 2 different ratios of β-TCP granules (10 and 24 w-% of β-TCP). The evaluation was realized in a long term rabbit bone model. After 24, 48 and 76 weeks, the implants were examined by micro CT, scanning electron microscopy (SEM) using backscattered electron (BSE) and light microscopy (polarized and blue light microscopy). No foreign body reaction was detected during the 76 weeks follow-up in any of the test samples. Polymer hydrolysis began at approximately 24 weeks, by 76 weeks, the pure polymer implant had begun to release P(96L/4D)LA particles and show signs of peripheral localized bone resorption. A decrease in the amount of CaP was noticed between 24 and 76 weeks in both 10 wt-% and 24 wt-% β-TCP/P(96L/4D)LA composites. The study showed that the highest bone in-growth was with 24 wt-% β-TCP/P(96L/4D)LA composite. Bone in-growth and mineralization were evident for the composites associated with specific peripheral bone architecture. Fluorescent labelling demonstrated high bone in-growth and remodeling at the interface, while for pure co-polymer no bone remodeling or bone activity was maintained after 48 weeks. The study demonstrated the positive effect of calcium phosphate content into P(96L/4D)LA. This kind of composite is a suitable resorbable osteoconductive matrix, which provides long term stability required for ligament fixation device.
Keywords: Co-polylactide/beta-tricalcium phosphate; Composite; Long term study; Bone regeneration;

A series of block poly(ester-urethane)s (abbreviated as PU3/4HB) based on biodegradable poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) segments were synthesized by a facile way of melting polymerization using 1,6-hexamethylene diisocyanate (HDI) as the coupling agent and stannous octanoate (Sn(Oct)2) as catalyst, with different 4HB contents and segment lengths. The chemical structure, molecular weight and distribution were systematically characterized by 1H nuclear magnetic resonance spectrum (NMR), Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC). The thermal property was studied by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The hydrophilicity was investigated by static contact angle of deionized water and CH2I2. DSC curves revealed that the PU3/4HB polyurethanes have their T g from −25.6 °C to −4.3 °C, and crystallinity from 2.5% to 25.3%, being almost amorphous to semi-crystalline. The obtained PU3/4HBs are hydrophobic (water contact angle 77.4°–95.9°), and their surface free energy (SFE) were studied. The morphology of platelets adhered on the polyurethane film observed by scanning electron microscope (SEM) showed that platelets were activated on the PU3/4HB films which would lead to blood coagulation. The lactate dehydrogenase (LDH) assay revealed that the PU3/4HBs displayed higher platelet adhesion property than raw materials and biodegradable polymer polylactic acid (PLA) and would be potential hemostatic materials. Crystallinity degree, hydrophobicity, surface free energy and urethane linkage content play important roles in affecting the LDH activity and hence the platelet adhesion. CCK-8 assay showed that the PU3/4HB is non-toxic and well for cell growth and proliferation of mouse fibroblast L929. It showed that the hydrophobicity is an important factor for cell growth while 3HB content of the PU3/4HB is important for the cell proliferation. Through changing the composition and the chain-length of P3/4HB-diol prepolymers, the biocompatibility of the poly(ester-urethane)s can be tailored.
Keywords: Block polyurethane; Biodegradable; Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)(P3/4HB); Synthesis; Characterization; Biocompatibility;

Stem cell-based therapy has been one of the best documented approaches in regenerative medicine, promising cures for a multitude of diseases and disorders. However, the ex vivo expansion of stem cells and their in vivo delivery are restricted by the limited availability of stem cell sources, the excessive cost of commercialization, and the anticipated difficulties of clinical translation and regulatory approval. An alternative to adoptively transferred stem cells are cell populations already present in a patient’s body, including stem/progenitor cells, which can be actively attracted to sites of injury. This technique, known as endogenous cell homing, has the potential to provide new therapeutic options for in situ tissue regeneration. Such options would be less costly and complex than approaches that require substantial ex vivo cell manipulation and that use artificial vehicles for cell delivery. Tissue regeneration methods that rely on endogenous stem/progenitor cell homing, local tissue responses, and functional stimulation thus offer new insights into in vivo tissue engineering and hold great promise for the future of translational medicine. Although such methods that take advantage of the latent endogenous regenerative potential of the patient are promising for the repair of damaged tissue, they are in need of further experimental support before application in late-stage diseases or severe tissue injury. This review is not meant to be exhaustive but gives a brief outlook on the promises, strategies, and current applications of endogenous stem cell homing for in situ tissue regeneration, with particular emphasis placed upon pharmacological means based on cell-instructive scaffolds and release technology to direct cell mobilization and recruitment. In the future these exciting paradigms are likely to help reconcile the clinical and commercial pressures in regenerative medicine.
Keywords: Cell homing; Tissue engineering; Endogenous regenerative technology; Biomimetic design; Drug delivery; Translational medicine;

Regulation of stem cell signaling by nanoparticle-mediated intracellular protein delivery by Dhiral A. Shah; Seok-Joon Kwon; Shyam S. Bale; Akhilesh Banerjee; Jonathan S. Dordick; Ravi S. Kane (3210-3219).
Intracellular delivery of specific proteins and peptides may be used to influence signaling pathways and manipulate cell function, including stem cell fate. Herein, we describe the delivery of proteins attached to hydrophobically modified 15-nm silica nanoparticles to manipulate specifically targeted cell signaling proteins. We designed a chimeric protein, GFP–FRATtide, wherein GFP acts as a biomarker for fluorescence detection, and FRATtide binds to and blocks the active site of glycogen synthase kinase-3β (GSK-3β) – a protein kinase involved in Wnt signaling. The SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, resulting in an elevation of β-catenin levels due to GSK-3β inhibition. Accumulation of β-catenin led to increased transcription of Wnt target genes, such as c-MYC, which instruct the cell to actively proliferate and remain in an undifferentiated state. The results presented here suggest that functional proteins can be delivered intracellularly in vitro using nanoparticles and used to target key signaling proteins and regulate cell signaling pathways. This ability is critical for the design of in vitro screens for gain/loss of pathway function, and may also prove to be useful for in vivo delivery applications.
Keywords: Protein delivery; Nanoparticle–protein conjugates; Wnt signaling; β-Catenin; GSK-3β; FRATtide;

A family of injectable and thermosensitive hydrogels suitable for myocardial injection was developed to deliver cardiosphere-derived cells (CDCs), an emerging and promising cell type for cardiac cell therapy. The hydrogels were based on polycaprolactone, N-isopropylacrylamide, 2-hydroxyethyl methacrylate and dimethyl-γ-butyrolactone acrylate. Atom transfer radical polymerization was used to synthesize hydrogels with a well-defined structure and well-controlled properties. The hydrogel solutions possessed thermal transition temperatures around room temperature and exhibited injectability suitable for myocardial injection. At 37 °C, the hydrogel solutions were capable of forming solid gels within 5s. This would allow the hydrogels to largely retain in the heart during injection. The hydrogels were highly flexible at body temperature with moduli matching those of the rat and human myocardium, and breaking strains higher than those of the myocardium, enabling them to respond synchronically with heart motion. The well-controlled polymer structure allowed for precisely controlling and decoupling water content and stiffness that affect cell differentiation. DNA assay demonstrated that CDCs proliferated in the 3D hydrogels during a 2-week culture period. CDCs maintained their colony formation capability in the hydrogel. Interestingly, hydrogels directed CDCs differentiation into mature cardiac lineage. At mRNA level, the mature cardiac specific transcript factors cardiac troponin T (cTnT) and cardiac myosin heavy chain (MYH6) were up-regulated, while the pre-mature cardiac marker GATA4 was down-regulated even after 1 day of encapsulation. CDC differentiation was interplayed by hydrogel stiffness and collagen in the hydrogel. Hydrogel with modulus ∼31 kPa was found to more significantly up-regulate cardiac expression than that with modulus ∼5 or ∼63 kPa. cTnT expression was largely regulated by both stiffness and collagen while MYH6 was mainly regulated by stiffness. Immunohistochemistry study showed that CDCs expressed cardiac troponin I and MYH6 proteins after 2 weeks of culture. These results demonstrate that the thermosensitive hydrogels not only possess physical properties suitable for myocardial injection, but also promote CDC proliferation and cardiac differentiation. These hydrogels represent potential candidates for delivery of CDCs into infarcted hearts.
Keywords: Poly(N-isopropylacrylamide) hydrogel; Cardiosphere-derived cell; Cell differentiation; Heart injection;

An overview of tissue and whole organ decellularization processes by Peter M. Crapo; Thomas W. Gilbert; Stephen F. Badylak (3233-3243).
Biologic scaffold materials composed of extracellular matrix (ECM) are typically derived by processes that involve decellularization of tissues or organs. Preservation of the complex composition and three-dimensional ultrastructure of the ECM is highly desirable but it is recognized that all methods of decellularization result in disruption of the architecture and potential loss of surface structure and composition. Physical methods and chemical and biologic agents are used in combination to lyse cells, followed by rinsing to remove cell remnants. Effective decellularization methodology is dictated by factors such as tissue density and organization, geometric and biologic properties desired for the end product, and the targeted clinical application. Tissue decellularization with preservation of ECM integrity and bioactivity can be optimized by making educated decisions regarding the agents and techniques utilized during processing. An overview of decellularization methods, their effect upon resulting ECM structure and composition, and recently described perfusion techniques for whole organ decellularization techniques are presented herein.
Keywords: Extracellular matrix; Biomaterials; Scaffolds; Decellularization; Regenerative medicine;

The promotion of neurological recovery in an intracerebral hemorrhage model using fibrin-binding brain derived neurotrophic factor by Qian Qian Han; Wei Jin; Zhi Feng Xiao; Jing Chun Huang; Hong Bin Ni; Jie Kong; Jun Wu; Bing Chen; Wei Bang Liang; Jian Wu Dai (3244-3252).
Brain derived neurotrophic factor (BDNF) has been shown to ameliorate recovery after intracerebral hemorrhage (ICH). The injured brain tissue after ICH is surrounded by hematoma formed from hemorrhage. Fibrin is abundant in hematoma, which could be a binding target for BDNF. In this work, we have fused a fibrin-binding domain (FBD) to BDNF (FBD-BDNF), and results demonstrate that FBD-BDNF has specific binding ability to fibrin and is retained in hematoma. Using the rat ICH model induced by bacterial collagenase, injected FBD-BDNF has been concentrated and retained at the hematoma. FBD has facilitated BDNF to exert targeting neuroprotective effect to the injured brain tissue around the hematoma after ICH. FBD-BDNF has significantly reduced the hemotoma volume, reduced tissue loss, promoted neural regeneration, and improved the rat behavioral performance.
Keywords: Brain; Nerve regeneration; Drug delivery; Wound healing;

The effect of acetylcholine-like biomimetic polymers on neuronal growth by Qin Tu; Li Li; Yanrong Zhang; Jianchun Wang; Rui Liu; Manlin Li; Wenming Liu; Xueqin Wang; Li Ren; Jinyi Wang (3253-3264).
Driven by clinical needs, nerve regeneration studies have recently become the focus of research and area of growth in tissue engineering. Biomimetic polymer synthesis and functional interface construction is a promising solution to induce neuritic sprouting and guide the regenerating nerve. However, few studies have been made on primary hippocampal neurons. In this study, a new type of acetylcholine-like biomimetic polymers for their potential in biomaterial-modulated nerve regeneration application is synthesized using click chemistry and free radical polymerization. The structure of the synthesized polymers includes a “bioactive” unit (acetylcholine-like unit) and a “bioinert” unit [poly(ethylene glycol) unit]. To explore the effects of the bioactive unit and the bioinert unit on neuronal growth, different ratios of the two initial monomers poly(ethylene glycol) monomethyl ether-glycidyl methacrylate (MePEG-GMA) and dimethylaminoethyl methacrylate (DMAEMA) were employed and five different polymers were synthesized. Their chemical structures were characterized using 1H nuclear magnetic resonance and Fourier-transform infrared spectroscopy, and their physical properties (including molecular weight, polydispersity, glass transition temperature, and melting point) were determined using gel permeation chromatography and differential scanning calorimetry. Culturing of the primary rat hippocampal neurons on the polymeric surfaces show that the ratio of the two initial monomers utilized for polymer synthesis significantly affects neuronal growth. Rat hippocampal neurons show different growth morphologies on different polymeric surfaces. The polymeric surface prepared with 1:60 (mol/mol) of MePEG-GMA to DMAEMA induces neuronal regenerative responses similar to that on poly-l-lysine, a very common benchmark material for nerve cell cultures. These results suggest that acetylcholine-like biomimetic polymers are potential biomaterials for neural engineering applications, particularly in modulating the growth of hippocampal neurons.
Keywords: Click chemistry; Free radical polymerization; Acetylcholine; Biomimetic material; Hippocampus neuron;

Single chain anti-c-Met antibody conjugated nanoparticles for in vivo tumor-targeted imaging and drug delivery by Ruei-Min Lu; Yu-Ling Chang; Min-Shan Chen; Han-Chung Wu (3265-3274).
Aberrantly expressed c-Met, the receptor for hepatocyte growth factor (HGF), has been implicated in human lung cancer as well as malignancy, metastasis and drug-resistance in other human cancers. Thus, this molecule could be a potential target for antibody-based cancer therapy. Targeting delivery of compound to tumor represented benefit for cancer detection and therapy. In this study, we utilized phage display to identify human single chain variable fragment (scFv) antibodies that specifically bound to c-Met protein. The anti-c-Met scFvs selectively bound to and internalized in several lung cancer cell lines expressing c-Met. Conjugation of anti-c-Met scFv with PEGylated liposomes enabled the efficient delivery of doxorubicin into cancer cells where it exerted cytotoxic activity by inducing apoptosis pathway. In addition, in vivo fluorescent imaging by scFv-conjugated quantum dots showed higher tumor uptake and increased tumor-normal tissue ratios. In a tumor xenograft model, anti-c-Met immunoliposome was found to selectively increase tumor accumulation of a chemotherapeutic drug and enhance its antitumor activity. Taken together, our results suggest that anti-c-Met scFv-mediated drug delivery systems show great promise in tumor-targeted therapy and imaging.
Keywords: c-Met; Phage display; scFv; Drug delivery system; Immunoliposome;

In vivo magnetic resonance imaging of cell tropsim, trafficking mechanism, and therapeutic impact of human mesenchymal stem cells in a murine glioma model by Li-Ying Chien; Jong-Kai Hsiao; Szu-Chun Hsu; Ming Yao; Chen-Wen Lu; Hon-Man Liu; Yao-Chang Chen; Chung-Shi Yang; Dong-Ming Huang (3275-3284).
Stem cells have offered much promise as delivery vehicles for brain tumor therapy, with the development of modalities to track the tumor tropism of stem cells receiving intense focus. Cellular magnetic resonance imaging (MRI) allows serial high-resolution in vivo detection of transplanted stem cells’ tropism toward gliomas in the mouse brain once these cells are internally labeled with iron oxide particles, but has been impeded by low labeling efficiencies. In this study, we describe the use of ferucarbotran and protamine (Fer-Pro) complexes for labeling human mesenchymal stem cells (hMSCs) for MRI tracking of glioma tropism in vivo. We found that Fer-Pro was not toxic and was highly efficient for labeling in vitro. Cell labeling with Fer-Pro promoted the migration of hMSCs toward glioma U87MG cells in vitro, which was mediated by stromal-derived factor-1/CXCR4 (SDF-1/CXCR4) signaling. Fer-Pro-labeled hMSCs could migrate specifically toward gliomas in vivo, which was observed with a clinical 1.5-T MRI system. The efficient labeling of Fer-Pro also allowed a tropic mechanism mediated by SDF-1/CXCR4 signaling to be detected by MRI in vivo. Additionally, the potential intrinsic inhibitory effect of hMSCs on glioma progression was estimated simultaneously. This is the first report to have used a clinical MRI modality to simultaneously study the migration, the therapeutic impact on tumors, and above all the trafficking mechanism of bone marrow-derived mesenchymal stem cells from human in a murine glioma xenograft model. The use of Fer-Pro for stem cell labeling may have potential clinical applications in stem cell guided therapy.
Keywords: Nanoparticle; MRI; Brain; Mesenchymal stem cell; Transplantation;

The antitumor efficacy of functional paclitaxel nanomicelles in treating resistant breast cancers by oral delivery by Hong-Juan Yao; Rui-Jun Ju; Xiao-Xing Wang; Yan Zhang; Ruo-Jing Li; Yang Yu; Liang Zhang; Wan-Liang Lu (3285-3302).
Paclitaxel has shown potent efficacy against a wide spectrum of cancers in clinical treatment. However, chemotherapy with paclitaxel has been limited due to serious allergic reactions in patients caused by cremophor EL, and multidrug resistance in many types of tumors, and the restricted permeability across the intestinal barrier. Functional paclitaxel nanomicelles were developed to overcome these obstacles. Evaluations were performed on the breast cancer MCF-7 and resistant MCF-7/Adr cells, MCF-7 and MCF-7/Adr tumor spheroids, Caco-2 cell manolayers, everted gut sacs and the xenografted resistant MCF-7/Adr cancers in nude mice. The functional paclitaxel nanomicelles were approximately of 15 nm in diameter, significantly increased the intracellular uptake of paclitaxel, and selectively accumulated into mitochondria and endoplasmic reticulum after treatment, showing strong inhibitory effect on MCF-7 and MCF-7/Adr cells. They were able to penetrate deeply into the central region of the MCF-7 and MCF-7/Adr spheroids, resulting in a significant reduction in the size of the spheroids. TEM observations showed that the intact functional paclitaxel nanomicelles were transported across the Caco-2 cell manolayer or the everted gut sac. A significant antitumor efficacy in the xenografted resistant MCF-7/Adr cancers in mice was evidenced by oral administration, which was comparable to intravenous administration. The functional paclitaxel nanomicelles would provide a strategy for oral administration of paclitaxel, increasing solubility of paclitaxel, and overcoming the multidrug resistant cancers.
Keywords: Functional paclitaxel nanomicelles; Oral administration; Transport across the intestinal barrier; Multidrug resistant; Breast cancer;

The structural orientation of antibody layers bound to engineered biosensor surfaces by Anton P. Le Brun; Stephen A. Holt; Deepan S.H. Shah; Charles F. Majkrzak; Jeremy H. Lakey (3303-3311).
This paper describes a membrane protein array that binds immunoglobulin G at its constant regions whilst leaving the variable regions free to bind antigen. The scaffold of the array is the transmembrane domain of outer membrane protein A (tOmpA) from Escherichia coli engineered to assemble as an oriented monolayer on gold surfaces via a single cysteine residue. Other protein domains can be fused to the N and C termini of the scaffold. In this study we use circularly permuted ctOmpA fused to two Z domains of Staphylococcus aureus protein A (ZZctOmpA) to create the immunoglobulin G-binding array. The solution structure of the engineered proteins was assessed by circular dichroism spectroscopy. Assembly of the array, attachment of antibodies and antigen binding were measured using surface plasmon resonance and neutron reflection. Compared to mouse IgG2, polyclonal IgG from rabbit bound very strongly to ZZctOmpA and the dissociation of the immunoglobulin was slow enough to allow neutron reflection studies of the assembled layer with antigen. Using both magnetic and isotopic contrasts a complete layer by layer model was defined which revealed that the 223 Å high layer contains antibodies in an upright orientation.
Keywords: Magnetism; Protein; Surface analysis; Surface modification; Gold; Immunochemistry;

In view of the prospective applications of polyamine coatings in functional gold nanoparticles for use as carriers in gene delivery systems, in tissue repair and as bactericidal and virucidal non-toxic vehicle, we have investigated the interactions of poly-l-lysine (PLL) with gold nanoparticles (AuNP). Since direct binding of PLL to AuNP is not strong at neutral pH, we have focused on PLL interactions with carboxylated self-assembled monolayers (SAM) on AuNP, such as the citrate-capped AuNP. The double-shell nanoparticles AuNP@Cit/PLL thus produced do not contain any toxic thiols. We have observed strong electrostatic interactions between polycationic chains of PLL and AuNP@Cit in weakly acidic to weakly alkaline solutions (pH 5–9), as evidenced by the bathochromic shift of the local surface plasmon (SP) band and strong increase in resonance elastic light scattering (RELS) intensity. The stoichiometry of interactions evaluated on the basis of RELS data indicates on a hyper-Langmuirian type of interactions with stoichiometric coefficient n = 1.35 (PLL : AuNP@Cit). From the RELS titration data, a shift of the deprotonation constant for the bound PLL has been determined (pK a = 11.6 for the bound PLL vs. 10.48 for the free PLL). The deprotonation of PLL leads to AuNP aggregate disassembly, evidenced by sharp RELS decline and hypsochromic shift of SP band. We have found that under these conditions, a residual aggregation due to the interparticle interactions between β-sheets of PLL overcoat become predominant. The molecular dynamics simulations indicate that multiple hydrogen bonds can also be formed between the PLL linker and the shell molecules of AuNP@Cit. The double-shell nanoparticles, AuNP@Cit/PLL, have been shown to attract DNA molecules using highly sensitive RELS measurements presenting the proof-of-concept for the suitability of this non-toxic nanostructured material for gene delivery applications. The advantage of the proposed material is no toxicity related to the ligand release in gene delivery processes in contrast to the thiol-functionalized AuNP.
Keywords: Poly-l-lysine coated nanoparticles; Gene delivery; Poly-l-lysine-mediated assembly; Au nanoparticles; RELS spectroscopy; Surface plasmon resonance;

Encapsulation of 2-methoxyestradiol within multifunctional poly(amidoamine) dendrimers for targeted cancer therapy by Yin Wang; Rui Guo; Xueyan Cao; Mingwu Shen; Xiangyang Shi (3322-3329).
We report here a general approach to using multifunctional poly(amidoamine) (PAMAM) dendrimer-based platform to encapsulate a potential anticancer drug for targeted cancer therapy. In this approach, amine-terminated generation 5 (G5) PAMAM dendrimers were sequentially modified with fluorescein isothiocyanate (FI) and folic acid (FA) via covalent conjugation, followed by an acetylation reaction to neutralize the remaining amines of the dendrimer surfaces. The synthesized multifunctional dendrimers (G5.NHAc-FI-FA) were then used to complex a potential anticancer drug, 2-methoxyestradiol (2-ME) for targeted delivery of the drugs to cancer cells overexpressing high-affinity folic acid receptors (FAR). We show that the formed G5.NHAc-FI-FA/2-ME complexes with each dendrimer encapsulating approximately 3.7 2-ME molecules are water soluble and stable. In vitro release studies show that 2-ME complexed with the multifunctional dendrimers can be released in a sustained manner. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in conjunction with cell morphology observation demonstrates that the G5.NHAc-FI-FA/2-ME complexes can specifically target and display specific therapeutic efficacy to cancer cells overexpressing high-affinity FAR. Findings from this study suggest that multifunctional dendrimers may be used as a general drug carrier to encapsulate various cancer drugs for targeted therapy of different types of cancer.
Keywords: Multifunctional dendrimers; 2-Methoxyestradiol; Encapsulation; Targeted cancer therapy;

We recently demonstrated that fibrin hydrogels can be used as vehicles for efficient lentivirus gene delivery. Gene transfer in fibrin gels was strongly dependent on matrix degradation by target cells but a fraction of lentiviral particles diffused out of the gels over time compromising spatial control of gene transfer. To overcome this challenge, we engineered lentiviral particles that bind covalently to fibrin during polymerization. To this end, we fused into the viral envelope glycoprotein (VSV-G) peptide domains that are recognized by factor XIII and protease cleavage sites that are recognized by plasmin. Lentivirus pseudotyped with the modified envelopes bound to fibrinogen in a factor XIII dose dependent manner and was released upon plasmin treatment. The peptide/VSV-G fusion envelope variants did not compromise the transduction efficiency of the resulting virus except when lacking any flexible linkers separating the peptide from the VSV-G envelope. Diffusion of virus from the gels decreased dramatically, especially at high concentrations of FXIII, even for fibrin gels with low fibrinogen concentration that were loaded with high titer virus. Lentivirus arrays prepared with fibrin-conjugated lentivirus yielded highly efficient gene transfer that was confined to virus-containing fibrin spots. As a result, signal/noise ratio increased and cross-contamination between neighboring sites was minimal. Finally, in addition to lentivirus microarrays this strategy may be used to achieve spatially-controlled gene transfer for therapeutic applications.
Keywords: Lentivirus; Genetically engineered envelope; Fibrin; Live cell array; Microarray;

Innovated catanionic solid lipid nanoparticles (CASLNs) carrying carmustine (BCNU) (BCNU-CASLNs) were grafted with anti-epithelial growth factor receptor (EGFR) (anti-EGFR/BCNU-CASLNs) and applied to inhibiting the propagation of human brain malignant glioblastomas cells. U87MG cells were treated with anti-EGFR/BCNU-CASLNs and stained for the expression of EGFR. The minimal average diameter of BCNU-CASLNs and maximal entrapment efficiency of BCNU emerged when the concentration of catanionic surfactants was 1 mm. An increase in the weight percentage of cacao butter (CB) reduced the zeta potential, enhanced the viability of human brain microvasscular endothelial cells (HBMECs), and decreased the expression of tumor necrosis factor-α by HBMECs. The dissolution rate of BCNU and inhibition against the multiplication of U87MG cells using anti-EGFR/BCNU-CASLNs followed the order: 100% CB > 0% CB > 50% CB. Anti-EGFR/BCNU-CASLNs demonstrated the properties including an effective delivery to U87MG cells and antiproliferative efficacy against the growth of malignant brain tumors.
Keywords: Catanionic surfactant; Solid lipid nanoparticle; Anti-epithelial growth factor receptor; Carmustine; Malignant glioblastomas cell;