Jiafu Cao

Size-controlled biodegradable nanoparticles: Preparation and size-dependent cellular uptake and tumor cell growth inhibition

Biodegradable nanoparticles with diameters below 1000nm are of great interest in the contexts of targeted delivery and imaging. In this study, we prepared PLGA nanoparticles with well-defined sizes of ∼70nm (NP70), ∼100nm (NP100), ∼200nm (NP200), ∼400nm (NP400), ∼600nm (NP600) and ∼1000nm (NP1000) using facile fabrication methods based on a nanoprecipitation and solvent evaporation techniques. The nanoparticles showed a narrow size distribution with high yield. Then the size-controlled biodegradable nanoparticles were used to investigate how particle size at nanoscale affects interactions with tumor cells and macrophages. Interestingly, an opposite size-dependent interaction was observed in the two cells. As particle size gets smaller, cellular uptake increased in tumor cells and decreased in macrophages. We also found that paclitaxel (PTX)-loaded nanoparticles showed a size-dependent inhibition of tumor cell growth and the size-dependency was influenced by cellular uptake and PTX release. The size-controlled biodegradable nanoparticles described in this study would provide a useful means to further elucidate roles of particle size on various biomedical applications.

Nitric oxide-releasing poly(lactic-co-glycolic acid)-polyethylenimine nanoparticles for prolonged nitric oxide release, antibacterial efficacy, and in vivo wound healing activity

Nitric oxide (NO)-releasing nanoparticles (NPs) have emerged as a wound healing enhancer and a novel antibacterial agent that can circumvent antibiotic resistance. However, the NO release from NPs over extended periods of time is still inadequate for clinical application. In this study, we developed NO-releasing poly(lactic-co-glycolic acid)-polyethylenimine (PEI) NPs (NO/PPNPs) composed of poly(lactic-co-glycolic acid) and PEI/diazeniumdiolate (PEI/NONOate) for prolonged NO release, antibacterial efficacy, and wound healing activity. Successful preparation of PEI/NONOate was confirmed by proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and ultraviolet/visible spectrophotometry. NO/PPNPs were characterized by particle size, surface charge, and NO loading. The NO/PPNPs showed a prolonged NO release profile over 6 days without any burst release. The NO/PPNPs exhibited potent bactericidal efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa concentration-dependently and showed the ability to bind on the surface of the bacteria. We also found that the NO released from the NO/PPNPs mediates bactericidal efficacy and is not toxic to healthy fibroblast cells. Furthermore, NO/PPNPs accelerated wound healing and epithelialization in a mouse model of a MRSA-infected wound. Therefore, our results suggest that the NO/PPNPs presented in this study could be a suitable approach for treating wounds and various skin infections.

Enzyme/pH dual sensitive polymeric nanoparticles for targeted drug delivery to the inflamed colon.

Novel nanoparticles whose drug release profiles are controlled by both enzyme and pH were prepared for the colon-specific drug delivery using a polymeric mixture of enzyme-sensitive azo-polyurethane and pH-sensitive Eudragit S100 (ES-Azo.pu). The enzyme/pH dual sensitive nanoparticles were designed to release a drug based on a two-fold approach which specifically aimed to target drug delivery to the inflamed colon while preventing the burst release of drugs in the stomach and small intestine. Single pH-sensitive (ES) and dual sensitive (ES-Azo.pu) nanoparticles were prepared using an oil-in-water emulsion solvent evaporation method and coumarin-6 (C-6) was used as a model drug. The successful formation of ES and ES-azo.pu nanoparticles that have 214 nm and 244 nm in mean particle size, respectively, was confirmed by scanning electron microscopy and qNano. ES nanoparticles showed almost 100% of burst drug release at pH 7.4, whereas ES-Azo.pu nanoparticles prevented the burst drug release at pH 7.4, followed by a sustained release phase thereafter. Furthermore, ES-Azo.pu nanoparticles exhibited enzyme-triggered drug release in the presence of rat cecal contents obtained from a rat model of colitis. An in vivo localization study in rat gastrointestinal tract demonstrated that ES-Azo.pu nanoparticles were selectively distributed in the inflamed colon, showing 5.5-fold higher C-6 than ES nanoparticles. In conclusion, the enzyme/pH dual sensitive nanoparticles presented in this study can serve as a promising strategy for colon-specific drug delivery against inflammatory bowel disease and other colon disorders.

Colon-targeted delivery of budesonide using dual pH- and time-dependent polymeric nanoparticles for colitis therapy

Single pH-dependent drug delivery systems have been widely used for colon-targeted delivery, but their efficiency is often hampered by the variation in gut pH. To overcome the limitation of single pH-dependent delivery systems, in this study, we developed and evaluated the therapeutic potential of budesonide-loaded dual pH/time-dependent nanoparticles (NPs) for the treatment of colitis. Eudragit FS30D was used as a pH-dependent polymer, and Eudragit RS100 as a time-dependent controlled release polymer. Single pH-dependent NPs (pH_NPs), single time-dependent NPs (Time_NPs), and dual pH/time-dependent NPs (pH/Time_NPs) were prepared using the oil-in-water emulsion method. The physicochemical properties and drug release profiles of these NPs in gastrointestinal (GI) tract conditions were investigated. The therapeutic potential and in vivo distribution of the NPs were evaluated in a dextran sulfate sodium (DSS)-induced colitis mice model. The pH/Time_NPs prevented a burst drug release in acidic pH conditions and showed sustained release at a colonic pH. The in vivo distribution study in the mice GI tract demonstrated that pH/Time_NPs were more efficiently delivered to the inflamed colon than pH_NPs were. Compared to the single pH_NPs-treated group, the pH/Time_NPs-treated group showed increased body weight and colon length and markedly decreased disease activity index, colon weight/length ratios, histological damage, and inflammatory cell infiltration in colon tissue. Our results demonstrate that the dual pH/time-dependent NPs are an effective oral colon-targeted delivery system for colitis therapy.

Phospho sulfonic acid: an efficient and recyclable solid acid catalyst for the solvent-free synthesis of α-hydroxyphosphonates and their anticancer properties

As a means of developing biologically active compounds, a series of α-hydroxyphosphonates, ArC(OH)PO(OR)2 (Ar = 5-G-2-OHPh; R = Me, Et, iPr, Bu; G = H, Br, Cl, NO2), have been synthesized by reacting diversely substituted salicylaldehydes with dialkyl phosphites by employing the Pudovik reaction in the presence of phospho sulfonic acid under neat conditions. The cytotoxicity of the compounds was tested in two human cancer cell lines by using MTT assay. Compounds bearing R = Et; G = Cl, R = Bu; G = Cl, and R = Et; G = NO2 showed good anticancer activity.

Herceptin-functionalized pure paclitaxel nanocrystals for enhanced delivery to HER2-postive breast cancer cells

The objective of this study was to prepare Herceptin (HCT)-functionalized paclitaxel nanocrystals and evaluated their cell-specific interactions, cellular accumulation, and growth inhibition in HER2-positve breast cancer cells as a tumor-targeted delivery module. Paclitaxel (PTX) was fabricated in the form of nanocrystals (PNCs) by a sono-precipitation method, and HCT were coated using a facile non-covalent method (PNCs-HCT). Our results showed that the PNCs-HCT were stable for at least 1month at 4°C with no noticeable desorption of HCT. The release test showed that PNCs-HCT exhibited sustained drug release similar to only PNCs but with a higher release rate than only PTX powder. Cellular uptake, cytotoxicity, and cell cycle arrest studies revealed that PNCs-HCT exhibit greater binding affinity and higher cell-specific internalization to HER2-positive breast cancer cell lines as compared to PNCs, followed by enhanced cell growth inhibition. HCT-functionalized PNCs presented in this study offer a promising strategy for targeted pure drug nanocrystal delivery and enhancing the efficiency of anticancer therapy.

Dexamethasone phosphate-loaded folate-conjugated polymeric nanoparticles for selective delivery to activated macrophages and suppression of inflammatory responses

Activated macrophages play a central role in the pathology of inflammatory diseases by secreting pro-inflammatory cytokines. In this study, we have developed folate-conjugated dexamethasone phosphate (DP)-loaded poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NP) that can selectively target to activated macrophages for alleviation of inflammatory responses. DP was loaded into PLGA nanoparticles in an amorphous form using an ionic interaction between DP, zinc, and PLGA. Folic acid (FA) was used as a targeting ligand for activated macrophages and conjugated onto DPNPs (FA-DPNPs). The mean particle size of DPNPs and FA-DPNPs was 126±36 and 133±28 nm, respectively. The zeta potential of DPNPs and FA-DPNPs were −13.5±0.4 mV and −14.9±0.9 mV, respectively. Sustained and controlled DP release over 48 hours from DPNPs and FA-DPNPs was observed. The low cellular uptake of both DPNPs and FA-DPNPs was observed in normal RAW264.7 macrophages, whereas significantly higher cellular uptake of FA-DPNPs was observed as compared to DPNPs in activated RAW264.7 macrophages. The production of pro-inflammatory cytokines (IL-6 and TNF-α) and nitric oxide (NO) from activated macrophages was inhibited more significantly by FA-DPNPs than by free DP and DPNPs. Taken together, DP-loaded nanoparticles, especially when conjugated with FA, were selectively internalized by activated macrophages and effectively suppressed inflammatory responses. DP loaded folate-conjugated PLGA nanoparticles presented in this study may offer a promising approach of targeted delivery to activated macrophages for the treatment of inflammatory diseases.

Enhanced therapeutic efficacy of budesonide in experimental colitis with enzyme/pH dual-sensitive polymeric nanoparticles.

Current colon-targeted drug-delivery approaches for colitis therapy often utilize single pH-triggered systems, which are less reliable due to the variation of gut pH in individuals and in disease conditions. Herein, we prepared budesonide-loaded dual-sensitive nanoparticles using enzyme-sensitive azo-polyurethane and pH-sensitive methacrylate copolymer for the treatment of colitis. The therapeutic potential of the enzyme/pH dual-sensitive nanoparticles was evaluated using a rat colitis model and compared to single pH-triggered nanoparticles. Clinical activity scores, colon/body weight ratios, myeloperoxidase activity, and proinflammatory cytokine levels were markedly decreased by dual-sensitive nanoparticles compared to single pH-triggered nanoparticles and budesonide solution. Moreover, dual-sensitive nanoparticles accumulated selectively in inflamed segments of the colon. In addition, dual-sensitive nanoparticle plasma concentrations were lower than single pH-triggered nanoparticles, and no noticeable in vitro or in vivo toxicity was observed. Our results demonstrate that enzyme/pH dual-sensitive nanoparticles are an effective and safe colon-targeted delivery system for colitis therapy.

The In Vitro and In Vivo Anti-Inflammatory Effects of a Phthalimide PPAR-γ Agonist

Previously, the authors found that 4-hydroxy-2-(4-hydroxyphenethyl) isoindoline-1,3-dione (PD1) (a phthalimide analogue) bound to and activated peroxisome proliferator-activated receptor-γ (PPAR-γ). Since PPAR-γ suppresses inflammatory responses, the present study was undertaken to investigate the anti-inflammatory effects of PD1. In lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages, PD1 suppressed the inductions of pro-inflammatory factors, including inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase 2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Concomitantly, PD1 enhanced the expressions of anti-inflammatory factors, such as arginase-1 and interleukin-10 (IL-10), and suppressed LPS-evoked nuclear factor kappa B (NF-κB) p65 subunit phosphorylation in macrophages. In addition, PPAR-γ activated by PD1 was intensively translocated to the nucleus. These observations suggest that the anti-inflammatory mechanism of PD1 involves inhibition of the NF-κB pathway. In a subsequent in vivo animal experiment conducted using a carrageenan-induced acute inflammatory rat paw edema model, intraperitoneal injection of PD1 significantly reduced paw swelling. Histological analysis of rat paw tissue sections revealed less infiltration of immune cells in PD1-pretreated animals. These findings suggest that PD1 be viewed as a lead compound for the development of novel anti-inflammatory therapeutics.

A three-dimensional assemblage of gingiva-derived mesenchymal stem cells and NO-releasing microspheres for improved differentiation

Stem cell therapy is an attractive approach to bone tissue regeneration. Nitric oxide (NO) has been reported to facilitate osteogenic differentiation of stem cells. To enhance osteogenic differentiation of gingiva-derived mesenchymal stem cells (GMSCs), we designed a method for in situ delivery of exogenous NO to these cells. A NO donor, polyethylenimine/NONOate, was incorporated into poly(lactic-co-glycolic acid) microspheres to deliver NO to the cells for an extended period of time under in vitro culture conditions. A hybrid aggregate of GMSCs and NO-releasing microspheres was prepared by the hanging drop technique. Confocal microscopy revealed homogeneous arrangement of the stem cells and microspheres in heterospheroids. Western blot analysis and live-dead imaging showed no significant change in cell viability. Importantly, the in situ delivery of NO within the heterospheroids enhanced osteogenic differentiation indicated by a 1.2-fold increase in alkaline phosphatase activity and an approximately 10% increase in alizarin red staining. In addition, a low dose of NO promoted proliferation of the GMSCs in this 3D system. Thus, delivery of the NO-releasing microsphers to induce differentiation of stem cells within this three dimensional system may be one of possible strategies to direct differentiation of a stem cell-based therapeutic agent toward a specific lineage.