Xing-Ping Qiu

Low molecular weight chitosan conjugated with folate for siRNA delivery in vitro: optimization studies

The low transfection efficiency of chitosan is one of its drawbacks as a gene delivery carrier. Low molecular weight chitosan may help to form small-sized polymer-DNA or small interfering RNA (siRNA) complexes. Folate conjugation may improve gene transfection efficiency because of the promoted uptake of folate receptor-bearing cells. In the present study, chitosan was conjugated with folate and investigated for its efficacy as a delivery vector for siRNA in vitro. We demonstrate that the molecular weight of chitosan has a major influence on its biological and physicochemical properties, and very low molecular weight chitosan (below 10 kDa) has difficulty in forming stable complexes with siRNA. In this study, chitosan 25 kDa and 50 kDa completely absorbed siRNA and formed nanoparticles (≤220 nm) at a chitosan to siRNA weight ratio of 50:1. The introduction of a folate ligand onto chitosan decreased nanoparticle toxicity. Compared with chitosan-siRNA, folate-chitosan-siRNA nanoparticles improved gene silencing transfection efficiency. Therefore, folate-chitosan shows potential as a viable candidate vector for safe and efficient siRNA delivery.

Phosphatase/temperature responsive poly(2-isopropyl-2-oxazoline)

We demonstrate a strategy for producing polymer bioconjugates which display both enzymatic and thermal responsiveness. This is achieved by combining the thermo-responsive properties of poly(2-isopropyl-2-oxazoline) (PiPrOx) with the self-assembly properties of fluorenylmethoxycarbonyl-tyrosine (Fmoc-Y), controlled by a phosphatase triggered mechanism.

New insights into the effects of molecular weight and end group on the temperature-induced phase transition of poly(N-isopropylacrylamide) in water

In an attempt to clarify issues related to the molecular weight dependence of the phase transition of poly(N-isopropylacrylamide) (PNIPAM) in water, we prepared a library of PNIPAM samples of well-controlled molecular weight (7000 to 45000 g/mol) bearing identical groups on each chain end. The polymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NIPAM) with a bifunctional chain tranfer agent and further end group modification. The effects of the end group chemical structure, hydroxyethyl (HE), propargyl (Pr), chloroethyl (CE), n-butyl (nBu), n-hexyl (nHe), and isobutylsulfanylthiosulfanyl (IBS) on the phase transition temperature of aqueous PNIPAM solutions were investigated by high-sensitivity differential scanning calorimetry (HS-DSC), yielding the enthalpy ΔH and the endotherm maximum temperature (TM), and turbidimetry, providing the cloud point (TCP) of each solution. The TCP and TM of the PNIPAM sample of lowest molar mass (Mn 7,000 g/mol, 0.5 g/L) ranged, respectively, from 38.8 to 22.5 °C and 42.2 to 26.0 °C, depending on the structure of the end-group, whereas ΔH showed no strong end-group dependence. The phase transition of all polymers, except α,ω-di(n-butyl-PNIPAM), exhibited a marked dependence on the polymer molar mass.

linear polyethylenimine produced by partial acid hydrolysis of poly(2-ethyl-2-oxazoline) for DNa and sirNa delivery in vitro

Polyethylenimines (PEIs) are the most efficient synthetic vectors for gene delivery available to date. With its high charge density and strong proton-buffering effect, PEI has an ability to condense DNA and small interfering RNA at physiologic pH. However, the polymer suffers from the disadvantage of high cellular toxicity. To reduce its cellular toxicity, we synthesized linear PEIs by partial hydrolysis of poly(2-ethyl-2-oxazoline). Three linear PEIs with different hydrolysis percentages (30%, 70%, and 96%, respectively) were produced as PEI30, PEI70, and PEI96. PEI30 and PEI96 cannot be considered as suitable transfection agents because of low transfection efficiency (PEI30) or high cellular toxicity (PEI96). PEI70 displayed very weak cell toxicity. The charge density of this polymer (PEI70) was strong enough to condense DNA and small interfering RNA at a physiologic pH of 7.4. Our results also show that PEI70 was highly efficient in DNA delivery and small interfering RNA-mediated knockdown of target genes. Thus, polymers such as PEI70 appear to be very promising vectors for gene delivery.

Hydrodynamic Delivery of Chitosan-Folate-DNA Nanoparticles in Rats with Adjuvant-Induced Arthritis

50 kDa chitosan was conjugated with folate, a specific tissue-targeting ligand. Nanoparticles such as chitosan-DNA and folate-chitosan-DNA were prepared by coacervation process. The hydrodynamic intravenous injection of nanoparticles was performed in the right posterior paw in normal and arthritic rats. Our results demonstrated that the fluorescence intensity of DsRed detected was 5 to 12 times more in the right soleus muscle and in the right gastro muscle than other tissue sections. β-galactosidase gene expression with X-gal substrate and folate-chitosan-plasmid nanoparticles showed best coloration in the soleus muscle. Treated arthritic animals also showed a significant decrease in paw swelling and IL-1β and PGE2 concentration in serum compared to untreated rats. This study demonstrated that a nonviral gene therapeutic approach using hydrodynamic delivery could help transfect more efficiently folate-chitosan-DNA nanoparticles in vitro/in vivo and could decrease inflammation in arthritic rats.

Synthesis of a poly(N-isopropylacrylamide) charm bracelet decorated with a photomobile α-cyclodextrin charm

Cyclic poly(N-isopropylacrylamide) with azobenzene inserted in the main chain (azo-c-PNIPAM, Mn 8800 g mol−1) was synthesized by the ‘click’ ring closure of α-azobenzene ω-azido poly(N-isopropylacrylamide) obtained by reverse addition–fragmentation transfer (RAFT) polymerization of NIPAM with an azobenzenyl-substituted chain transfer agent. Taking advantage of the azobenzene–α-cyclodextrin inclusion complex and conducting the ring closure in the presence of excess α-cyclodextrin (α-CD), we prepared azo-c-PNIPAM with an interlocked α-CD of topology reminiscent of a single-charm bracelet. The polymers were characterized by gel permeation chromatography, 1H NMR spectroscopy, UV-visible absorption spectroscopy, and FTIR spectroscopy. 2D NOESY spectroscopy confirmed that the azobenzene group of the ring was inserted into the α-CD cavity. Trans to cis isomerization of the azobenzene group was induced by irradiation at 365 nm. The cis-azobenzene moiety was expelled from the α-CD cavity, as indicated by a hypsochromic shift of the π–π* electronic transition, corroborated by circular induced dichroism (CD) spectroscopy, 1H NMR and 2D NOESY spectroscopy. The reverse cis to trans isomerization induced by irradiation at 440 nm triggered the re-insertion of the azobenzene group into the α-CD cavity.

Efficient Nonviral Gene Therapy Using Folate-Targeted Chitosan-DNA Nanoparticles In Vitro

Nonviral cationic polymers like chitosan can be combined with DNA to protect it from degradation. The chitosan is a biocompatible, biodegradable, nontoxic, and cheap polycationic polymer with low immunogenicity. The objective of this study was to synthesize and then assess different chitosan-DNA nanoparticles and to select the best ones for selective in vitro transfection in human epidermoid carcinoma (KB) cell lines. It revealed that different combinations of molecular weight, the presence or absence of folic acid ligand, and different plasmid DNA sizes can lead to nanoparticles with various diameters and diverse transfection efficiencies. The intracellular trafficking, nuclear uptake, and localization are also studied by confocal microscopy, which confirmed that DNA was delivered to cell nuclei to be expressed.

Temperature-Dependent Adsorption and Adsorption Hysteresis of a Thermoresponsive Diblock Copolymer

A nonionic-cationic diblock copolymer, poly(2-isopropyl-2-oxazoline)60-b-poly((3-acrylamidopropyl)trimethylammonium chloride)17, (PIPOZ60-b-PAMPTMA17), was utilized to electrostatically tether temperature-responsive PIPOZ chains to silica surfaces by physisorption. The effects of polymer concentration, pH, and temperature on adsorption were investigated using quartz crystal microbalance with dissipation monitoring and ellipsometry. The combination of these two techniques allows thorough characterization of the adsorbed layer in terms of surface excess, thickness, and water content. The high affinity of the cationic PAMPTMA17 block to the negatively charged silica surface gives rise to a high affinity adsorption isotherm, leading to (nearly) irreversible adsorption with respect to dilution. An increase in solution pH lowers the affinity of PIPOZ to silica but enhances the adsorption of the cationic block due to increasing silica surface charge density, which leads to higher adsorption of the cationic diblock copolymer. Higher surface excess is also achieved at higher temperatures due to the worsening of the solvent quality of water for the PIPOZ block. Interestingly, a large hysteresis in adsorbed mass and other layer properties was observed when the temperature was cycled from 25 to 45 °C and then back to 25 °C. Possible causes for this temperature hysteresis are discussed.

Synthesis and Association Behavior of Telechelic Poly(N-isopropylacrylamides) with Azobenzene End Groups

A telechelic poly(N-isopropylacrylamide) (PNIPAM) with azobenzene end-groups (telechelic Az-PNIPAM) was prepared by reversible addition-fragmentation chain transfer (RAFT) free radical polymerization of NIPAM. This polymer self-assembles in cold water forming nanoparticles with a hydrodynamic radius (R H) of 8 nm and an aggregation number of 29 chains. The thermoresponsive behavior of Az-PNIPAM in water was investigated by turbidimetry and light scattering. In addition, the photoresponsive behavior of aqueous Az-PNIPAM was investigated by monitoring the changes with temperature of the solution and transmittance at 650 nm before and after irradiation at 366 nm.

Spreading of Cell Aggregates on Zwitterion-Modified Chitosan Films

The sulfobetaine (SB) moiety, which comprises a quaternary ammonium group linked to a negatively charged sulfonate ester, is known to impart nonfouling properties to interfaces coated with polysulfobetaines or grafted with SB-polymeric brushes. Increasingly, evidence emerges that the SB group is, overall, a better antifouling group than the phosphorylcholine (PC) moiety extensively used in the past. We report here the synthesis of a series of SB-modified chitosans (CH-SB) carrying between 20 and 40 mol % SB per monosaccharide unit. Chitosan (CH) itself is a naturally derived copolymer of glucosamine and N-acetyl-glucosamine linked with a β-1,4 bond. Analysis by quartz crystal microbalance with dissipation (QCM-D) indicates that CH-SB films (thickness ∼ 20 nm) resist adsorption of bovine serum albumin (BSA) with increasing efficiency as the SB content of the polymer augments (surface coverage ∼ 15 μg cm–2 for films of CH with 40 mol % SB). The cell adhesivity of CH-SB films coated on glass was assessed by determining the spreading dynamics of CT26 cell aggregates. When placed on chitosan films, known to be cell-adhesive, the CT26 cell aggregates spread by forming a cell monolayer around them. The spreading of CT26 cell aggregates on zwitterion-modified chitosans films is thwarted remarkably. In the cases of CH-SB30 and CH-SB40 films, only a few isolated cells escape from the aggregates. The extent of aggregate spreading, quantified based on the theory of liquid wetting, provides a simple in vitro assay of the nonfouling properties of substrates toward specific cell lines. This assay can be adopted to test and compare the fouling characteristics of substrates very different from the chemical viewpoint.