Shanfeng Wang

Targeted Delivery of Gemcitabine to Pancreatic Adenocarcinoma Using Cetuximab as a Targeting Agent

One of the key challenges in anticancer therapy is the toxicity and poor bioavailability of the anticancer drugs. Nanotechnology can play a pivotal role by delivering drugs in a targeted fashion to the malignant cells that will reduce the systemic toxicity of the anticancer drug. In this report, we show a stepwise development of a nanoparticle-based targeted delivery system for in vitro and in vivo therapeutic application in pancreatic cancer. In the first part of the study, we have shown the fabrication and characterization of the delivery system containing gold nanoparticle as a delivery vehicle, cetuximab as a targeting agent, and gemcitabine as an anticancer drug for in vitro application. Nanoconjugate was first characterized physico-chemically. In vitro targeting efficacy, tested against three pancreatic cancer cell lines (PANC-1, AsPC-1, and MIA Paca2) with variable epidermal growth factor receptor (EGFR) expression, showed that gold uptake correlated with EGFR expression. In the second part, we showed the in vivo therapeutic efficacy of the targeted delivery system. Administration of this targeted delivery system resulted in significant inhibition of pancreatic tumor cell proliferation in vitro and orthotopic pancreatic tumor growth in vivo. Tumor progression was monitored noninvasively by measuring bioluminescence of the implanted tumor cells. Pharmacokinetic experiments along with the quantitation of gold both in vitro and in vivo further confirmed that the inhibition of tumor growth was due to targeted delivery. This strategy could be used as a generalized approach for the treatment of a variety of cancers characterized by overexpression of EGFR.

Physical Properties and Cellular Responses to Crosslinkable Poly(Propylene Fumarate)/Hydroxyapatite Nanocomposites

A series of crosslinkable nanocomposites has been developed using hydroxyapatite (HA) nanoparticles and poly(propylene fumarate) (PPF). PPF/HA nanocomposites with four different weight fractions of HA nanoparticles have been characterized in terms of thermal and mechanical properties. To assess surface chemistry of crosslinked PPF/HA nanocomposites, their hydrophilicity and capability of adsorbing proteins have been determined using static contact angle measurement and MicroBCA protein assay kit after incubation with 10% fetal bovine serum (FBS), respectively. In vitro cell studies have been performed using MC3T3-E1 mouse pre-osteoblast cells to investigate the ability of PPF/HA nanocomposites to support cell attachment, spreading, and proliferation after 1, 4, and 7 days. By adding HA nanoparticles to PPF, the mechanical properties of crosslinked PPF/HA nanocomposites have not been increased due to the initially high modulus of crosslinked PPF. However, hydrophilicity and serum protein adsorption on the surface of nanocomposites have been significantly increased, resulting in enhanced cell attachment, spreading, and proliferation after 4 days of cell seeding. These results indicate that crosslinkable PPF/HA nanocomposites are useful for hard tissue replacement because of excellent mechanical strength and osteoconductivity.

Photo-cross-linked hybrid polymer networks consisting of poly(propylene fumarate) and poly(caprolactone fumarate): controlled physical properties and regulated bone and nerve cell responses.

Aiming to achieve suitable polymeric biomaterials with controlled physical properties for hard and soft tissue replacements, we have developed a series of blends consisting of two photo-cross-linkable polymers: polypropylene fumarate (PPF) and polycaprolactone fumarate (PCLF). Physical properties of both un-cross-linked and UV cross-linked PPF/PCLF blends with PPF composition ranging from 0% to 100% have been investigated extensively. It has been found that the physical properties such as thermal, rheological, and mechanical properties could be modulated efficiently by varying the PPF composition in the blends. Thermal properties including glass transition temperature (T g) and melting temperature (T m) have been correlated with their rheological and mechanical properties. Surface characteristics such as surface morphology, hydrophilicity, and the capability of adsorbing serum protein from culture medium have also been examined for the cross-linked polymer and blend disks. For potential applications in bone and nerve tissue engineering, in vitro cell studies including cytotoxicity, cell adhesion, and proliferation on cross-linked disks with controlled physical properties have been performed using rat bone marrow stromal cells and SPL201 cells, respectively. In addition, the role of mechanical properties such as surface stiffness in modulating cell responses has been emphasized using this model blend system.

Photo-crosslinked poly(ε-caprolactone fumarate) networks for guided peripheral nerve regeneration: Material properties and preliminary biological evaluations

In an effort to achieve suitable biomaterials for peripheral nerve regeneration, we present a material design strategy of combining a crystallite-based physical network and a crosslink-based chemical network. Biodegradable polymer disks and conduits have been fabricated by photo-crosslinking three poly(epsilon-caprolactone fumarate)s (PCLF530, PCLF1250, and PCLF2000), which were synthesized from the precursor poly(epsilon-caprolactone) (PCL) diols with nominal molecular weights of 530, 1250, and 2000 g mol(-1), respectively. Thermal properties such as glass transition temperature (T(g)), melting temperature (T(m)), and crystallinity of photo-crosslinked PCLFs were examined and correlated with their rheological and mechanical properties. Furthermore, in vitro degradation of uncrosslinked and crosslinked PCLFs in PBS crosslinked PCLFs in 1 N NaOH aqueous solution at 37 degrees C was studied. In vitro cytocompatibility, attachment, and proliferation of Schwann cell precursor line SPL201 cells on three PCLF networks were investigated. Crosslinked PCLF2000 with the highest crystallinity and mechanical properties was found to best support cell attachment and proliferation. Using a new photo-crosslinking method, single-lumen crosslinked PCLF nerve conduits without defects were fabricated in a glass mold. Crosslinked PCLF2000 nerve conduits were selected for evaluation in a 1cm gap rat sciatic nerve model. Histological evaluation demonstrated that the material was biocompatible with sufficient strength to hold sutures in place after 6 and 17 weeks of implantation. Nerve cable with myelinated axons was found in the crosslinked PCLF2000 nerve conduit.

Heparin-immobilized polymers as non-inflammatory and non-thrombogenic coating materials for arsenic trioxide eluting stents.

We have synthesized heparin-immobilized copolymers of L-lactide (LA) and 5-methyl-5-benzyloxycarbonate-1,3-dioxan-2-one (MBC) as non-inflammatory and non-thrombogenic biodegradable coating materials. These copolymers were used in fabricating arsenic trioxide (As(2)O(3))-eluting stents to reduce the late-stage adverse events, such as thrombosis, localized hypersensitivity and inflammation, that occur when applying stents to treat coronary artery diseases. Heparinized copolymers effectively reduced platelet adhesion and protein adsorption while increasing the plasma recalcification time and thromboplastin time in vitro. Histological analysis of the polymer-coated stents in a porcine coronary artery injury model indicated that one heparinized copolymer (Hep-Co90, LA:MBC=90:10), with the highest LA content of 90% and the lowest degradation rate, induced the least foreign body reactions and inflammation, which were as small as those induced by bare metal stents. Consequently, Hep-Co90 was used as the stent coating material for local As(2)O(3) delivery. Histomorphometric evaluations suggested no significant difference between bare metal stents and As(2)O(3)-eluting stents at 1 and 3 months post-implantation. At 6 months, the lumen area in the porcine coronary arteries treated with As(2)O(3)-eluting stents is 32.4% higher than those treated with bare metal stents while the neointimal area, neointimal thickness and stenosis rate decreased by 25.8%, 32.5% and 31.2%, respectively. The As(2)O(3)-eluting stent using Hep-Co90 as the drug carrier and stent coating material presented in this study represents a novel promising device in preventing in-stent restenosis.

Enhanced Cell Ingrowth and Proliferation through Three-Dimensional Nanocomposite Scaffolds with Controlled Pore Structures

We present enhanced cell ingrowth and proliferation through cross-linked three-dimensional (3D) nanocomposite scaffolds fabricated using poly(propylene fumarate) (PPF) and hydroxyapatite (HA) nanoparticles. Scaffolds with controlled internal pore structures were produced from computer-aided design (CAD) models and solid freeform fabrication (SFF) technique, while those with random pore structures were fabricated by a NaCl leaching technique for comparison. The morphology and mechanical properties of scaffolds were characterized using scanning electron microscopy (SEM) and mechanical testing, respectively. Pore interconnectivity of scaffolds was assessed using X-ray microcomputed tomography (micro-CT) and 3D imaging analysis. In vitro cell studies have been performed using MC3T3-E1 mouse preosteoblasts and cultured scaffolds in a rotating-wall-vessel bioreactor for 4 and 7 days to assess cell attachment, viability, ingrowth depth, and proliferation. The mechanical properties of cross-linked nanocomposite scaffolds were not significantly different after adding HA or varying pore structures. However, pore interconnectivity of PPF/HA nanocomposite scaffolds with controlled pore structures has been significantly increased, resulting in enhanced cell ingrowth depth 7 days after cell seeding. Cell attachment and proliferation are also higher in PPF/HA nanocomposite scaffolds. These results suggest that cross-linked PPF/HA nanocomposite scaffolds with controlled pore structures may lead to promising bone tissue engineering scaffolds with excellent cell proliferation and ingrowth.

Parabolic dependence of material properties and cell behavior on the composition of polymer networks via simultaneously controlling crosslinking density and crystallinity.

A systematic investigation was performed on regulating materials properties and cell behavior using hybrid networks composed of amorphous poly(propylene fumarate) (PPF) and three poly(epsilon-caprolactone) diacrylates (PCLDAs) with variance in crystallinity and melting temperature. Through controlling both crosslinking density and crystallinity in the photo-crosslinked PPF/PCLDA blends, mechanical properties could be tuned efficiently in a wide range. For PCLDA synthesized from a low-molecular weight PCL diol precursor with a low crystallinity and a low melting point, crosslinks could completely suppress crystalline domains over the composition range in the PPF/PCLDA networks. Consequently, tensile, shear, torsional, and compression moduli all increased with the composition of PPF or the crosslinking density continuously for amorphous PPF/PCLDA networks. For PCLDAs synthesized using two PCL diols with higher molecular weights, crystallinity remained for the PCLDA compositions between approximately 80% and 100%. Minimum moduli and tensile stress at break were found at the lowest required composition of PPF for suppressing crystallinity. Surface physicochemical properties and morphology of the crosslinked blend disks have been characterized and their capabilities of adsorbing proteins from cell culture medium have been determined. Using both mouse MC3T3-E1 cells and rat Schwann cell precursor line (SpL201) cells, cell responses to these polymer networks such as cell adhesion, spreading, and proliferation were found to be dramatically distinct on different polymer networks and demonstrated non-monotonic or parabolic dependence on the network composition, coincident with the composition dependence of the mechanical properties.

Distinct cell responses to substrates consisting of poly(ε-caprolactone) and poly(propylene fumarate) in the presence or absence of cross-links.

To investigate the role of chemical cross-links in regulating biomaterial properties and cell behavior, we have prepared and characterized a series of biodegradable polymer blends in both un-cross-linked and photo-cross-linked forms. In this comparative study, these blends consisted of an oligomeric, cross-linkable, amorphous poly(propylene fumarate) (PPF) and a high-molecular-weight, semicrystalline poly(ε-caprolactone) (PCL). After cross-linking, semi-interpenetrating polymer networks (semi-IPNs) were formed by combining PPF chemical network and PCL physical network that was associated by the crystallites. The material design strategy presented here was different from previously studied semicrystalline polymer networks, in which crystallizable segments participated covalently in the chemical network and were significantly suppressed by the network. For these PPF/PCL blends, thermal properties such as melting temperature (T(m)) and crystallinity have been correlated with their rheological and mechanical properties to demonstrate the effects of cross-linking density and crystallinity. Surface morphology, hydrophilicity, and the capability of adsorbing proteins from cell culture media have also been determined. For potential applications in bone and vascular tissue engineering and demonstration of regulating cell behavior on polymer substrates with controllable physicochemical characteristics, in vitro cell studies that included cell viability, attachment, spreading, and proliferation have been performed using mouse MC3T3 cells and primary rat aortic smooth muscle cells (SMCs). In a similar manner, these two cell types have been found to show distinct cell responses to the polymer substrates in the presence or absence of cross-links.

Exposed hydroxyapatite particles on the surface of photo-crosslinked nanocomposites for promoting MC3T3 cell proliferation and differentiation

We present a systematic study for investigating the role of exposed hydroxyapatite (HA) nanoparticles in influencing surface characteristics and mouse pre-osteoblastic MC3T3-E1 cell behavior using nanocomposites prepared by photo-crosslinking poly(ε-caprolactone) diacrylate (PCLDA) with HA. PCLDA530 and PCLDA2000 synthesized from poly(ε-caprolactone) diol precursors with nominal molecular weights of 530 and 2000 g mol(-1) were used as the polymer matrices. Crosslinked PCLDA530 was amorphous while crosslinked PCLDA2000 was semi-crystalline. Crosslinked PCLDA/HA composites with different compositions of HA (10%, 20% and 30%) as well as crosslinked PCLDAs were characterized in terms of their composition-dependent physicochemical properties. The tensile, compressive and shear moduli were greatly enhanced by incorporating HA nanoparticles with the polymer matrices. The disk surfaces of original crosslinked PCLDA/HA nanocomposites were removed by cutting using a blade to expose HA nanoparticles that were embedded in the polymer substrates. The composition of HA was much higher on the cut surface, particularly in semi-crystalline crosslinked PCLDA2000/HA nanocomposites. The surface characteristics of original and cut crosslinked PCLDA/HA nanocomposites were compared and correlated with cell behavior on these nanocomposites. MC3T3-E1 cell attachment, proliferation and differentiation were significantly enhanced when the HA composition was increased in original crosslinked PCLDA/HA nanocomposites due to more bioactive HA, higher surface stiffness and rougher topography. More exposed HA on the surface of cut semi-crystalline PCLDA2000/HA nanocomposites resulted in improved hydrophilicity and significantly better MC3T3 cell attachment, proliferation and differentiation compared with the original surfaces. This study suggests that HA nanoparticles may not be fully exploited in polymer/HA nanocomposites where the top polymer surface covers the particles. The removal of this polymer layer can generate more desirable surfaces and osteoconductivity for bone repair and regeneration.

Poly(ethylene glycol)-grafted poly(propylene fumarate) networks and parabolic dependence of MC3T3 cell behavior on the network composition

We present a method to modify poly(propylene fumarate) (PPF), an injectable biomaterial for bone-tissue-engineering applications, by photo-crosslinking it with methoxy poly(ethylene glycol) monoacrylate (mPEGA) at various mPEGA compositions of 0-30%. The bulk properties such as thermal and rheological properties of uncrosslinked mPEGA/PPF blends and the mechanical properties of photo-crosslinked mPEGA/PPF blends were also investigated and correlated with surface characteristics to elaborate on the modulation of mouse MC3T3 cell adhesion, spreading, proliferation and differentiation through controlled physicochemical properties. Unlike PPF crosslinked with PEG dimethacrylate, mPEGA chains tethered on the surface of crosslinked PPF did not influence the swelling ratio in water while increased surface hydrophilicity greatly. Meanwhile, surface frictional coefficient and the capability of adsorbing proteins from cell culture medium decreased continuously with increasing the mPEGA composition in mPEGA/PPF networks. Demonstrating cell repulsive effect at the mPEGA compositions higher than 7%, the modified surfaces improved MC3T3 cell attachment, proliferation and differentiation, which reached maxima at the mPEGA composition of 5-7%. Besides revealing that mPEGA pendant chains could enhance cell responses by increasing hydrophilicity when their fraction on the hydrophobic surface was small, the present study also offered a new method of improving the wettability and performance of the scaffolds made from PPF for bone repair.