Synthesis of folate modified chitosan-based nanomicelles and its anti-tumor activity / 浙江大学学报·医学版

OBJECTIVE@#To design and synthesize folate-modified pH-responsive chitosan-based nanomicelles and investigate the anti-tumor activity of the drug-loaded micelles.@*METHODS@#CHI-DMA was obtained by reductive amination reaction of aldehyde-based chitosan and hydrophilic amine compounds, and CHI-DMA-LA was obtained by condensation reaction with lauric acid; FA-CHI-DMA-LA was obtained after modification with folic acid (FA). The drug-loaded nanomicelles FA-CHI-DMA-LA/DOX were assembled by solvent change method. The physicochemical properties of polymers were characterized by hydrogen nuclear magnetic resonance and transmission electron microscope. The particle size and surface potential were determined by dynamic light scattering method. Folic acid access rate, doxorubicin (DOX) loading rate and entrapped efficiency were measured by UV-vis spectrophotometer. The drug release properties of DOX-loaded micelles were monitored by fluorescence spectrophotometer at different pHs (7.4, 6.5, 5.0). The cytotoxicity against human oral cancer KB cells was detected by MTT assay. Fluorescence microscope and flow cytometry were applied to investigate the phagocytosis of DOX-loaded micelles on KB cells.@*RESULTS@#FA-CHI-DMA-LA was synthesized. The particle sizes of FA-CHI-DMA-LA-1 and FA-CHI-DMA-LA-2 micelles which used for the subsequent experiments were (73±14) nm and (106±15) nm, zeta potential were (15.59±1.98) mV and (21.20±2.35) mV, respectively. The drug loading rates of drug-loaded micelles FA-CHI-DMA-LA-1/DOX and FA-CHI-DMA-LA-2/DOX are (4.08±1.12)%and (4.12±0.44)%, respectively. drug release is pH-responsive, with cumulative release of DOX up to 37%and 36%at pH 5.0, which is about 1.5 times higher than that of pH 7.4. For FA-CHI-DMA-LA micelles with 1.25 to 125 μg/mL concentration, the survival rate of KB cells is more than 70%after incubation for 24 hours. The cell uptake of FA-CHI-DMA-LA/DOX micelles was enhanced compared to CHI-DMA-LA/DOX, and the cell uptake was higher in incubation without FA medium than that with FA. Compared with free DOX or CHI-DMA-LA/DOX, FA-CHI-DMA-LA/DOX nanomicelles showed higher cyctoxicity to KB cells, especially the FA-CHI-DMA-LA-2/DOX nanomicelles, the cell survival rate was about 17% after incubation for 24 hours.@*CONCLUSIONS@#FA-modified chitosan-based nanomicelle with good biocompatibility was successfully prepared, which exhibits tumor microenvironmental pH responsive drug release and tumor targeting.

Solvent assisted size effect on AuNPs and significant inhibition on K562 cells.

Herein, the synthesis and characterization of ideal size (∼10 and 40 nm, in diameter) AuNPs (gold nanoparticles) were reported. Two different organic solvents such as DMF (dimethyl formamide) and NMPL (N-methyl-2-pyrrolidone) were used to synthesize AuNPs along with agents reducing agents such as NaBH4 (sodium borohydrate) and Na3C6H5O7 (sodium citrate). The combination of [(HAuCl4)-(DMF)-(NaBH4)] gives AuNPs with an avg. size of 10.2 nm. Similarly, the combination of [(HAuCl4)-(NMPL)-(Na3C6H5O7)] gives AuNPs with an avg. size of 40.4 nm. The morphology of these nanoscale AuNPs has been characterized through TEM and HRTEM imaging followed by SAED for lattice parameters such as d-spacing value (2.6 Å/0.26 nm) of crystalline metal (Au) nanoparticles. Further, these unique and ideal nanoscale AuNPs were used to evaluate the potential working efficacy by using in vitro cell based studies on K562 (leukaemia) blood cancer cells. From the MTT assay results around 88% cell inhibition was measured for ∼10 nm sized AuNPs. The treated cells were stained with different fluorescent dyes such as FITC, DAPI, Rho-6G and their ruptured morphology has been reported in the respective sections. These types of ideal sized metal (Au) nanoparticles are recommended for various theranostics such as to cure breast, colon, lung and liver cancers.

Synthesis and cell biological properties of polyaspartic acid drug/gene vector / 浙江大学学报·医学版

OBJECTIVE: Taking polysuccinimide as the main chain, amine side chain and alkyl side chain were grafted to prepare the drug/gene co-delivery vector. The property of the polymers with various side links were investigated to select an optimal vector. METHODS: Poly-D, L-polysuccinimide was synthesized by polymerization reaction of D, L-aspartic acid as monomer. Therefore, N, N-dimethylenedipropyl-triamine and 3, 3'-diaminodipropylamine were grafted with dodecylamine/adecylamine/octadecylamine at different proportions by ring-opening reaction to obtain amphiphilic PEECs. The structure of the material was confirmed by 1H NMR; the particle size and surface potential of the micelles were measured by dynamic light scattering; the critical micelle concentration (CMC) was determined by pyrene fluorescent probe; the RNA blocking ability was characterized by agarose gel electrophoresis; the release behavior of the PEECs was examined and the cytotoxicity, cellular uptake and gene silencing efficiency of the PEECs were studied at the cellular level. RESULTS A series of PEECs with different grafting rates was successfully synthesized. The particle sizes and surface potential of the PEEC derived micelles were between 250 nm and 350 nm and 27 mV and 45 mV, respectively, with a small CMC value. The RNA binding ratio of PEECs was at a mass ratio of about 0.8:1. MTT assay demonstrated that PEEC micelles had certain cytotoxicity. PEECs had excellent micelle formation, drug-loading and gene binding abilities, particularly, PEEC16-2 showed high gene silencing efficiency at the cellular level. CONCLUSIONS PEECs are able to co-delivery drug and gene, and PEEC16-2 micelles have the best ability of drug encapsulation and gene delivery.

[Synthesis, characterization and antitumor activity of 5-fluorouracil-nicotinamide cocrystal].

Objective: To synthesize 5-fluorouracil-nicotinamide (5-FU-NCT) cocrystal and to investigate its physicochemical and biological properties. Methods: The cocrystal of 5-Fu-NCT was prepared through the cooling technology. PXRD, NMR, FTIR and DSC were used to characterize the structure of 5-FU-NCT cocrystal. Solubility was measured by HPLC method. Drug resistant human liver cancer BEL-7402/5-FU cells were treated with 5-FU-NCT cocrystal, the inhibition effect was tested by MTT and HE staining, and cancer cell migration was determined by scratch test. Results: According to PXRD, NMR, FTIR and DSC results, the cocrystal of 5-Fu-NCT had been synthesized successfully. The characteristic diffraction peaks (2θ/°) of the cocrystal were 16.4, 20.4, 22.3, 27.9 and 30.1. The solubility of 5-FU-NCT was 13.5 g/L as measured by HPLC. The antitumor activity tests showed that 5-FU-NCT cocrystal enhanced anticancer effect of 5-FU, and the IC50 of 5-FU and 5-FU-NCT was 129.6 µg/mL and 42.6 µg/mL, respectively. Conclusion: 5-Fu-NCT cocrystal have been synthesized successfully through the cooling technology and it shows an enhanced anticancer effect in comparison to 5-FU on BEL-7402/5-FU cells.

[Preparation, characterization and cytology study of Pluronic-PEI micelles].

Objective: To prepare and characterize Pluronic-PEI micelles as a drug/gene delivery system. Methods: We used the low-molecular-weight PEI as a cross-linking agent to prepare the Pluronic-PEI micelles. The particle size, zeta potential and critical micelle concentration (CMC) were measured by dynamic light scattering (DLS) and pyrene fluorescence probe. The cytotoxicity, transfection efficiency and the impact on the intracellular ATP and P-gp levels of Pluronic-PEI micelles were investigated at the cellular level. Results: Pluronic-PEI micelles were successfully prepared with a suitable particle size (120-180 nm), zeta potential (+6-+9 mv), and a good ability to carry the drug/gene. An in-vitro study showed that Pluronic-PEI had low cytotoxicity, and the P123-PEI600 possessed high gene transfection efficiency and could downregulate the intracellular ATP and P-gp levels. Conclusion: Pluronic-PEI is a good drug/gene delivery system, and P123-PEI600 is an ideal vector, which may be used in the combination therapy for reversing multidrug resistance.

[Synthesis of BODIPY photosensitizers and their photodynamic effect on cancer cells].

Objective: To design and synthesize photosensitizers with different substituents and to identify its physicochemical characteritics and photodynamic effect on cancer cells. Methods: Two kinds of BODIPY photosensitizers BPOI and BPCI were synthesized through condensation reaction between aldehyde and reactive hydrogen of pyrrole, followed with electrophilic substitution reaction. Physicochemical properties were characterized by 1H NMR, FT-IR and UV-visible absorption spectra and fluorescence emission spectra. The ability to produce reactive oxygen species was detected by BPDF and DCFH-DA. Photodynamic therapy effect on rat glioma C6 cells in vitro was determined by MTT method. Results: Two kinds of BODIPY photosensitizers BPOI and BPCI were successfully synthesized with different substituents, which were confirmed by 1H NMR, FT-IR. Both materials had low toxicity and could be readily taken up by tumor cells. The ability of synthesized photosensitizers to produce reactive oxygen species was strongly influenced by solvent polarity when the substituent was electron-donating group, while no effect was found when the substituent was electron-withdrawing group. Conclusion: Photosensitizer BPOI with electron-donating substituent produces reactive oxygen species with a slow rate in a highly polar environment, while greatly enhanced this effect in a low polarity environment, which is expected to be used for environmental-selective photodynamic therapy in tumor cells.

[Bacterial outer membrane vesicles as nano carriers to study immunological activities].

Objective: To prepare a nano-carrier based on combining bacterial outer membrane vesicles (OMV) with three block polymer pluronic F127 (PEO100-PPO65-PEO100) (OMV-F127) and to investigate its immunological activity. Methods: Attenuated salmonella (sal) was cultivated. OMV were separated by centrifugal ultrafiltration or ultrasonication, and OMV-F127 was prepared by mechanical extrudation method. The protein contents and compositions were tested with BCA and SDS-PAGE; the morphology of OMV, F127 and OMV-F127 were observed with FM and TEM; the particle sizes and their zeta potential were determined with DLS. Mouse macrophage RAW246.7 cells were treated with OMV-F127 (50 µg/mL, 100 µg/mL) in vitro, and the concentrations of IL-12, TNF-α and IFN-γ in culture supernatant were measured with ELISA kits. Results: The contents of protein in separated OMV by centrifugal ultrafiltration and ultrasonication were 2.8 mg/mL and 2.7 mg/mL, respectively. SDS-PAGE showed the marker protein OmpF/C in OMV. Under the FM and TEM, ball-like structure of F127 and OMV-F127 was observed. Size analysis revealed that the diameters of OMV, F127 and OMV-F127 were 72±2 nm, 90±3 nm and 92±2 nm, respectively. ELISA tests revealed that OMV-F127 significantly stimulated the secretion of IL-12, TNF-α and IFN-γ in RAW246.7 cells. Conclusion: A nano-carrier based on bacterial outer membrane vesicles has been prepared, which can stimulate the secretion of cytokines and may have immunomodulatory effects.

[Preparation, characterization and antitumor of cyclodextrin inclusion of an anti-cancer drug regorafenib].

Objective: In order to improve the drug's solubility, dissolution and bioavailability, RG-ß-CD, RG-γ-CD and RG-Hp-ß-CD were prepared by co-crystallization between Regorafenib (RG) and ß-cyclodextrin (ß-CD), γ-cyclodextrin (γ-CD) and Hydroxypropyl-ß-cyclodextrin (Hp-ß-CD). Methods: Three inclusion complexes were prepared by recrystallization and solvent evaporation methods and characterized by fourier transform infrared spectroscopy (FT-IR), thermal analysis (TG), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), 1H nuclear magnetic resonance (1H-NMR), nuclear overhauser effect spectroscopy (NOESY). In vivo experiments, tumor suppression assay were made with SW620 colon cancer cell. Results: The ability of solubility and dissolution were improved after inclusion with three kinds of cyclodextrins. The regorafenib-ß-cyclodextrin inclusionis proved to have the best stability. The less enhanced was regorafenib-γ-cycl-odextrin inclusion. The best dissolution of regorafenib-ß-cyclodextrin inclusion complex was to bring as the tumor suppression assay, the result shows that regorafenib inclusion with ß-cyclodextrin is better than regorafenib itself. Conclusion: The bioavailability of regorafenib by inclusion with cyclodextrin can enhance due to the solubility enhancement of RG, which can provide an effective method for improving solubility and dissolution of insoluble drug in clinical medication.

[Characterization and stability of S (-) pantoprazole sodium hydrates].

Objective: To study the characteristics and stability of new S(-) pantoprazole sodium hydrates. Methods: The X-ray single crystal diffractometer (SXRD), X-ray powder diffractometer (PXRD), thermogravimetric analysis (TG) and infrared spectrometry (IR) were used to characterize S(-) pantoprazole sodium hydrates. The stability of the hydrates was evaluated by high temperature test,affecting factors test and accelerated test. Results: The crystalline water in S(-) pantoprazole sodium hydrates were very easy to lose and obtain, but crystal structure was not changed significantly. The transition from S(-) pantoprazole sodium trihydrate to S(-) pantoprazole sodium hemipentahydrate occurred at approximately 40 ℃ and reversible transitions from hemipentahydrate to trihydrate occurred at high humidity. Two hydrates had no significant difference in accelerated test. Conclusion: The crystal structure of the two hydrates are almost the same, hemipentahydrate is more stable than trihydrates at high temperature or at exposure to light(at 4500 ± 500 lx).

[Advances in nanoparticle-targeting tumor associated macrophages for cancer imaging and therapy].

Tumor tissues are composed of tumor cells and complicate microenvironment. Tumor associated macrophages (TAMs) as an important component in tumor microenvironment, play fundamental roles in tumor progression, metastasis and microenvironment regulation. Recently, studies have found that nanotechnology, as an emerging platform, provides unique potential for cancer imaging and therapy. With the nanotechnology, TAMs imaging presents direct evidence for cancer development, progression, and the effectiveness of cancer treatments; it also can regulate the immunosuppression of tumor microenvironment and improve therapeutic efficiency through TAMs targeted killing or phenotypic transformation. In this article, we illustrate the function of TAMs and review the latest development in nano-carriers and their applications in tumor associated macrophage targeting cancer imaging and therapy.

Synthesis, characterization and antitumor activity of 5-fluorouracil-nicotinamide cocrystal / 浙江大学学报·医学版

To synthesize 5-fluorouracil-nicotinamide (5-FU-NCT) cocrystal and to investigate its physicochemical and biological properties. The cocrystal of 5-Fu-NCT was prepared through the cooling technology. PXRD, NMR, FTIR and DSC were used to characterize the structure of 5-FU-NCT cocrystal. Solubility was measured by HPLC method. Drug resistant human liver cancer BEL-7402/5-FU cells were treated with 5-FU-NCT cocrystal, the inhibition effect was tested by MTT and HE staining, and cancer cell migration was determined by scratch test. According to PXRD, NMR, FTIR and DSC results, the cocrystal of 5-Fu-NCT had been synthesized successfully. The characteristic diffraction peaks (2θ/°) of the cocrystal were 16.4, 20.4, 22.3, 27.9 and 30.1. The solubility of 5-FU-NCT was 13.5 g/L as measured by HPLC. The antitumor activity tests showed that 5-FU-NCT cocrystal enhanced anticancer effect of 5-FU, and the IC50 of 5-FU and 5-FU-NCT was 129.6 μg/mL and 42.6 μg/mL, respectively. 5-Fu-NCT cocrystal have been synthesized successfully through the cooling technology and it shows an enhanced anticancer effect in comparison to 5-FU on BEL-7402/5-FU cells.

Alpha,beta-poly(N-hydroxypropyl/aminoethyl)-DL-aspartamide -co-L-lysine: potential non-viral vehicle for gene delivery / 生物医学工程学杂志

A series of Poly[aspartic acid-co-L-lysine](PAL) are copolycondensed by DL-aspartice acid and L-lysine with different ratios. Their constructions are identified by the spectra of 1H-NMR, FT-IR, X-Ray). These spectra are proved to have good regularity of these copolymers. alpha,beta-Poly[(N-hydroxypropyl/aminoethyl)-DL-Aspartamide-co-L-lysine] (PHAAL) is synthesized by ring-opening poly [aspartic acid-co-lysine] (PAL). PHAAL has good degradability in the phosphoric acid buffer solution (0.01 M, pH = 7.4) in the enzyme solution (Papain, Trypsine). PHAAL appeared tobe low cytotoxicity in Hela, ECV-304, Bcap37 cell lines, which was quantified by MTT assay. The combination ability of PHAAL with plasmid DNA was evaluated by agarose gel electrophoresis with agarose gel (1.0% w/v) containing ethidium bromide (0.25 microg/ml). The PHAAL with higher ratios of lysine in the copolymers have higher ability of condensing DNA. In summary, PHAAL, the polyaminoacid materials, could be one kind of macromolecule materials tobeused as the non-viral gene vehicle.

A new biodegradable poly-amino acid: alpha,beta-poly[(N-hydroxypropyl/aminoethyl)-DL-aspartamide-co-L-lysine], a potential nonviral vector for gene delivery.

A new class of biodegradable poly-amino acid, alpha,beta-poly[(N-hydroxypropyl/aminoethyl)-DL-Aspartamide-co-L-Lysine] (PHAAL), was synthesized by ring-opening of poly[succinimide-co-lysine](PSL) with n-propanolamine and ethylene diamine after thermal copolycondensation of DL-Aspartic acid and L-lysine under reduced pressure. Different ratio feeds of PSL were obtained and characterized by 1H-NMR, Fourier transformed infrared spectroscopy, X-ray, thermogravimetric analysis and gel permeation chromatography experiments. As one of the polycationic materials, performed for gene delivery carrier, the PHAAL degradation experiment was carried out in PBS (10 mM, pH =7.4) and enzyme (papain, trypsine 1 mg/ml, 37 +/- 0.1 degree C) solution. PHAAL had lower cytotoxicity than polyethylenimine (25KDa) and poly-L-Lysine (30 KDa), in Hela, E.C.V.-304, Bcap 37 cell lines. Particle size and zeta, potential of PHAAL/DNA complexes were measured. Sizes ranged from 300-500 nm and zeta potentials were at -20 to 2,5 mV. The condensation ability of PHAAL for DNA was evaluated by agarose gel electrophoresis. The PHAAL could completely neutralize DNA at N/P ratio (w/w) 150:1.

Synthesis, characterization and in vitro release of poly (succinimide-co-4-aminobutanoic acid) by acid-catalyzed polycondensation of L-aspartic acid and 4-aminobutanoic acid / 生物医学工程学杂志

For the purpose of increasing the hydrophilicity of poly aspartic acid, a series of polymer of L-aspartic acid and 4-aminobutanoic acid with different ratios (mol/mol) were prepared. The copolymers were characterized by 13CNMR, DSC and x-ray. The confirmed the structures of the polymers. In-vitro tests of release at phosphate buffer saline, enzyme solution of trypsin and papain (37.0 degrees C, pH = 7.4) were carried out. The result indicated that the polymers could be degraded in some degree, and that 4-aminobutanoic acid segments accelerated the degradation rate of the polymers. Skin irritation test and systemic acute toxicity test were carried out, which showed that the polymer was a nontoxic biomedical material.