The Co-Delivery of Oxaliplatin Abrogates the Immunogenic Response to PEGylated siRNA-Lipoplex

Purpose

In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex.

Methods

Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated.

Results

Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses.

Conclusions

Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.     

全反式维甲酸提高人结肠癌LoVo细胞对奥沙利铂敏感性

目的探讨全反式维甲酸(all-trans retinoic acid,AT-RA)提高人结肠癌LoVo细胞对奥沙利铂的药物敏感性和机制。方法MTT法筛选ATRA和奥沙利铂实验浓度。流式细胞仪检测ATRA对细胞周期的影响。分别用ATRA、奥沙利铂、ATRA联合奥沙利铂作用LoVo细胞;MTT法检测药物抑制率,流式细胞仪检测细胞周期及凋亡率,原子光谱吸收仪检测细胞DNA含铂(Pt)量。结果奥沙利铂抑制LoVo细胞作用呈量效依赖;其GI50(GI,inhibit net cell growth by%)为6.5mg.L-1,主要阻滞肿瘤细胞在S期。ATRA抑制LoVo细胞作用呈时效和量效依赖。1.0μmol.L-1ATRA作用12h后G1期细胞增多;48h后伴S期、G2/M期细胞减少并明显抑制肿瘤细胞增殖;作用12h至48h后联合奥沙利铂,两药联合由相加作用转变为协同作用;联合用药后S期细胞明显增多,细胞DNA含Pt量明显增加,但不提高奥沙利铂凋亡率。结论小剂量ATRA通过改变LoVo细胞周期和提高细胞DNA含Pt量,明显增加肿瘤细胞对奥沙利铂的药物敏感性。     

Fucosylated Multiwalled Carbon Nanotubes for Kupffer Cells Targeting for the Treatment of Cytokine-Induced Liver Damage

Purpose

To develop, characterize and exploring the sulfasalazine loaded fucoyslated multi walled carbon nanotubes for Kupffer cell targeting for effective management of cytokine-induce liver damage.

Methods

Sulfasalazine was loaded into the fucosylated MWCNTs after subsequential functionalization (carboxylation, acylation and amidation) using dialysis membrane technique. The in vitro, in vivo studies were performed on macrophages J 774 cell line for Kupffer cells targeting for the treatment of cytokine-induced liver damage.

Results

The loading of SSZ into SSZ-FUCO-MWCNTs was 87.77?±?0.11% (n?=?3). Sustained release was obtained from SSZ-FUCO-MWCNTs, with 89.12?±?0.71% of SSZ released into medium at 48th hr. SSZ-FUCO-MWCNTs showed the 9.01?±?0.23% hemolysis was drastically reduced from 21.62?±?0.24% SSZMWCNTs 21.62?±?0.24%. In SRB assay, SSZ-FUCO-MWCNTs showed more cytotoxicity than raw and SSZ-MWCNTs. In cytokine assay, SSZ- FUCO-MWCNTs exhibited significantly higher inhibition of IL-12 p40 secretion. In Western blot assay, SSZ-FUCO-MWCNTs significantly inhibit NF-κB activation.

Conclusion

The results suggested that the SSZ-FUCO-MWCNTs may be useful nano-carriers for targeted delivery to Kupffer cells in the treatment of cytokine-induced liver damage.     

Evaluation of Antiproliferative Activity,Safety and Biodistribution of Oxaliplatin and 5-Fluorouracil Loaded Lactoferrin Nanoparticles for the Management of Colon Adenocarcinoma: an <Emphasis Type="BoldItalic">In Vitro</Emphasis> and an <Emphasis Type="BoldItalic">In Vivo</Emphasis> Study

Purpose

Colon adenocarcinoma is the most common form of gastro intestinal tract cancer, predominantly in ageing population. Chemotherapy with 5-Fluorouracil and oxaliplatin is an indispensable treatment regimen, nevertheless having limitation of systemic toxicity and lower therapeutic index. The present study is based on evaluation of anti-proliferative potential, pharmacokinetics parameters, safety profile, biodistribution and efficacy of 5-FU/oxaliplatin loaded lactoferrin nanoparticles in cell lines and wistar rats in order to overcome the above limitation.

Methods

Nanoparticles were prepared by Water-in-oil process. The anti-proliferative efficacy and mode of cellular entry was evaluated in COLO-205 cells. The pharmacokinetics and biodistribution analysis were performed in healthy rats while efficacy and safety assay were performed in ACF induced rats.

Results

5-FU and oxaliplatin loaded nanoparticles shows enhanced antiproliferative activity as compare to free drugs in COLO-205 cells. Lactoferrin nanoparticles also improve the pharmacokinetics profile, safety parameters and efficacy of 5-FU and Oxaliplatin.

Conclusion

Lactoferrin nanoparticles demonstrated an attractive drug delivery module to manage the colon adenocarcinoma as it has improved the antiproliferative activity of 5-FU and Oxaliplatin against colon adenocarcinoma cells. Moreover, it also improves the pharmacokinetic profile and safety parameters of the same drug in wistar rat.

     

Is an Alternative Drug Delivery System Needed for Docetaxel? The Role of Controlling Epimerization in Formulations and Beyond

Purpose

The presence of 7-epidocetaxel in docetaxel injection and in vivo epimerisation has been reported to be the cause for development of tumor resistance to chemotherapy including docetaxel by inducing tumor cell protein cytochrome P450 1B1. The objective of this study was to determine systemic toxicity of Taxotere® containing 10% 7-epidocetaxel and to develop PEGylated liposomal injection that could resist epimerization in vivo. Another need for PEGylated liposomal delivery of docetaxel is to avoid reported hypersensitivity reactions of marketed products like Taxotere® and Duopafei® containing high concentration of tween-80.

Methods

The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. The in vivo toxicity of Taxotere® containing 10% 7-epimer was studied in B16F10 experimental metastasis model.

Results

B16F10 experimental metastasis model using C57BL/6 mice injected with Taxotere® containing 10% 7-epimer showed higher weight loss as compared to Taxotere® containing no epimer at single dose of 40 mg/kg indicating higher systemic toxicity. Incubation of PEGylated liposomes with phosphate buffer saline (pH 7.4) containing 0.1% w/v Tween-80 for 48 h showed better resistance to docetaxel degradation when compared with Taxotere® injection indicating better in vivo stability of liposomal docetaxel. In addition, PEGylated liposomes showed enhanced in vitro cytotoxicity, against A549 and B16F10 cells, than Taxotere®.

Conclusion

We can therefore expect less in vivo conversion of liposomal loaded docetaxel into 7-epimer, more passive targeting to tumor tissues, decreased 7-epimer induced systemic toxicity and tumor resistance to chemotherapy compared to Taxotere®. Further in vivo studies are needed to ascertain these facts.     

Nanoparticles Based on a Hydrophilic Polyester with a Sheddable PEG Coating for Protein Delivery

Purpose

To investigate the effect of polyethylene glycol (PEG) in nanoparticles based on blends of hydroxylated aliphatic polyester, poly(D,L-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and PEG-PLGHMGA block copolymers on their degradation and release behavior.

Methods

Protein-loaded nanoparticles were prepared with blends of varying ratios of PEG-PLGHMGA (molecular weight of PEG 2,000 and 5,000 Da) and PLGHMGA, by a double emulsion method with or without using poly(vinyl alcohol) (PVA) as surfactant. Bovine serum albumin and lysozyme were used as model proteins.

Results

PEGylated particles prepared without PVA had a zeta potential ranging from ~ ?3 to ~?35 mV and size ranging from ~200 to ~600 nm that were significantly dependent on the content and type of PEG-block copolymer. The encapsulation efficiency of the two proteins however was very low (<30%) and the particles rapidly released their content in a few days. In contrast, all formulations prepared with PVA showed almost similar particle properties (size: ~250 nm, zeta potential: ~?1 mV), while loading efficiency for both model proteins was rather high (80–90%). Unexpectedly, independent of the type of formulation, the nanoparticles had nearly the same release and degradation characteristics. NMR analysis showed almost a complete removal of PEG in 5 days which explains these marginal differences.

Conclusions

Protein release and particle degradation are not substantially influenced by the content of PEG, likely because of the fast shedding of the PEG blocks. These PEG shedding particles are interesting system for intracellular delivery of drugs.     

A Novel Three-Dimensional Large-Pore Mesoporous Carbon Matrix as a Potential Nanovehicle for the Fast Release of the Poorly Water-soluble Drug,Celecoxib

Purpose

A novel mesocellular carbon foam (MSU-FC) with a large pore size and a three-dimensional porous structure for the oral delivery of poorly water-soluble drugs was prepared. The goal of this study was to improve in vitro dissolution and in vivo absorption of celecoxib (CEB), a model drug, by means of novel carbon-based nanoparticles prepared from the MSU-FC matrix.

Methods

The MSU-FC matrix was synthesized by an inverse replica templating method using mesocellular silica template. A solvent immersion/evaporation method was used to load the drug molecules. The drug-loaded nanoparticles were characterized for morphology, surface area, particle size, mesoporous structure, crystallinity, solubility and dissolution. The effect of MSU-FC on cell viability was measured using the MTT conversion assay. Furthermore, the oral bioavailability of CEB-loaded MSU-FC in fasted rats was compared with that of the marketed product.

Results

Our results demonstrate that CEB incorporation into the prepared MSU-FC resulted in an approximately 9-fold increase in aqueous solubility in comparison with crystalline CEB. MSU-FC produced accelerated immediate release of CEB in comparison with crystalline CEB (pure CEB powder or marketed formulation) and the drug-loaded conventional mesoporous carbon particles. The relative bioavailability of CEB for CEB-loaded MSU-FC was 172%. In addition, MSU-FC nanoparticles exhibited very low toxicity.

Conclusions

The MSU-FC nanomatrix has been shown to be a promising drug delivery vehicle for improving the dissolution and biopharmaceutical characteristics of poorly water-soluble drugs.     

Development of High-Content Gemcitabine PEGylated Liposomes and Their Cytotoxicity on Drug-Resistant Pancreatic Tumour Cells

Purpose

The objective of this study was to develop high-content gemcitabine PEGylated liposomes to reverse gemcitabine resistance in pancreatic tumour cells. The mechanism of drug loading into liposomes was also investigated.

Methods

To increase the drug entrapment efficiency (EE) and drug loading (DL), a novel passive loading approach named Small Volume Incubation method (SVI) was developed and compared to the reverse phase evaporation (REV) and remote loading methods. The in vitro cytotoxicity was evaluated using MIA PaCa-2 and Panc-1 cell lines.

Results

The EE for remote loading was 12.3?±?0.3%, much lower than expected and a burst release was observed with the resultant liposomes. Using the optimized SVI method, increased EE (37?±?1%) and DL (4%, w/w) were obtained. The liposomes (200?±?5 nm) showed minimal drug leakage, good stability, and significant improvement in cytotoxicity to the gemcitabine-resistant pancreatic cancer cell lines.

Conclusions

Remote loading was not suitable for loading gemcitabine into liposomes. pK_a?>?4.6 for basic drugs and intra-liposomal precipitation of loaded compounds were suggested as an additional requirement to the current criteria for remote loading using ammonium sulphate gradient (pK_a?相似文献   

Pharmacokinetics of cisplatin during hyperthermic intraperitoneal treatment of peritoneal carcinomatosis

Purpose

Cisplatin during hyperthermic intraperitoneal chemotherapy (HIPEC) has not previously been measured with a selective technique. The primary aims were to examine the pharmacokinetics of active cisplatin and its monohydrated complex (MHC) during HIPEC using a specific measuring technique, to compare cisplatin’s systemic absorption with oxaliplatin, and to compare active cisplatin levels to that of total platinum.

Methods

Ten patients treated with cytoreductive surgery and HIPEC (cisplatin 50 mg/m²,doxorubicin 15 mg/m²) were recruited. Blood and perfusate samples were drawn during and after HIPEC. Cisplatin analysis was conducted using liquid chromatography (LC) with post-column derivatization with diethyldithiocarbamate and compared with inductively coupled plasma-mass spectrometry (ICP-MS).

Results

The mean half-life (t1/2) of perfusate cisplatin was 18.4 min, with area under the time-concentration curve (AUC) 0–90 min of 2.87 mM·min and estimated 0–60 min of 2.45 mM·min. The absorption t1/2 was 9.0 min for cisplatin and 18.2 min for oxaliplatin. The ratio of total platinum to active cisplatin increased in a linear manner by time of perfusion.

Conclusions

Cisplatin is absorbed quicker than oxaliplatin. Lowering the perfusion time to 60 min does not significantly change the pharmacokinetics of cisplatin, and is therefore to be considered. As the HIPEC perfusion progresses, the ICP-MS technique does not adequately reflect active cisplatin levels in the perfusate.     

BSA-PLGA-Based Core-Shell Nanoparticles as Carrier System for Water-Soluble Drugs

Purpose

Preparation, optimization and in vitro evaluation of core-shell nanoparticles comprising of a hydrophilic core of BSA surrounded by a hydrophobic shell of PLGA for loading water-soluble drugs.

Methods

A double emulsion method was optimized for preparation of BSA-PLGA based core-shell nanoparticles. Proof of concept for core-shell type structure was established by visual techniques like confocal microscopy and TEM. Characterization was done for particle size, encapsulation efficiency, drug loading and in vitro drug release. Cellular uptake was assessed using confocal microscopy, bio-TEM and HPLC assay, and cytotoxic activity was tested by MTT assay in MG-63 osteosarcoma cells.

Results

The optimized core-shell nanoparticles showed a particle size of 243 nm (PDI-0.13) and encapsulation efficiency of 40.5% with a drug loading of 8.5% w/w. In vitro drug release studies showed a sustained release for 12 h. Cellular uptake studies indicated a rapid and efficient uptake within 2 h. TEM studies indicated that the core-shell nanoparticles were localized in cytoplasm region of the cells. Gemcitabine loaded core-shell nanoparticles showed enhanced cytotoxicity against MG-63 cells as compared to marketed formulation of gemcitabine (GEMCITE®).

Conclusion

These results indicate that core-shell nanoparticles can be a good carrier system for delivering hydrophilic drugs like gemcitabine successfully to the cells with enhanced efficacy.

The Shape/Morphology Balance: A Study of Stealth Liposomes via Fractal Analysis and Drug Encapsulation

Purpose

Fractal analysis was used as a tool in order to study the morphological characteristics of PEGylated liposomes. We report on the morphological characteristics of stealth liposomes composed of DPPC and DPPE-PEG 3000 in two dispersion media using fractal analysis.

Methods

Light scattering techniques were used in order to elucidate the size, the morphology and the surface charge of PEGylated liposomes as a function of PEGylated lipid concentration and temperature. Fluorescence spectroscopy studies revealed a microenvironment of low polarity inside the liposomal membranes.

Results

All formulations were found to retain their physicochemical characteristics for at least 3 weeks. The hydrodynamic radii (R_h) of stealth liposomes were stable in the process of heating up to 50°C; while the fractal dimension values (d_f) which correspond to their morphology, have been changed during heating. Hence, these results are a first indication of the presence of a heterogeneous microdomain structure of the stealth liposomal system. The amphiphilic drug indomethacin (IND) was successfully encapsulated within the liposomes and led to an increased size of stealth liposomes, while the morphology of liposomal vectors changed significantly at the highest molar ratio of PEGylated lipid.

Conclusions

We can state that this approach can promote a new analytical concept based on the morphological characteristics and quantify the shape of drug carriers complementary to that of the conventional analytical techniques.     

Morphological Modification of Carbon Nanoparticles after Interacting with Methotrexate as a Potential Anticancer Agent

Purpose

Production of highly penetrable and targetable drug delivery particles is mainly focused by current therapy and such focus is achieved in our present study. The carbon nanoparticle (CNP) prepared from purely natural source was modified from spherical shape to cylindrical floral like structure after treatment with the anticancer drug methotrexate (CM).

Methods

The physiochemical properties of the CNP and CM was characterized using FT-IR/Raman Spectrometer, XRD, SEM, AFM, particle size analyzer and its biological evaluation using haemolysis and MTT assay.

Results

The shift in FT-IR peaks at 1592, 1120 cm^?1 and peaks of raman spectra observed at 1303, 1300 cm^?1 represents ordered carbon nanotubes. The morphological change from spherical to cylindrical floral like structure was observed using SEM and AFM and its particle size distribution analysis shows an average diameter of 269 nm for CM. XRD peak at 2θ?=?23.86° (002) indicates the presence of large amount of amorphous material that corresponds to multi-walled carbon nanotubes. Haemocompatibility studies proved the safety level usage as 100 μg/ml and MTT assay shows viability rate of 85–98% with mouse embryonic fibroblast (NIH/3 T3) and 30–45% with pancreatic carcinoma (MIA PaCa-2) and gastric cancer cell lines (SNU- 484) respectively.These results are also supported by phase contrast microscope images observed after staining with calcein AM and EthD-1.

Conclusions

The morphologically modified CNPs has shown good anticancer, biocompatibility and haemocompatibility property which is an important criterion to be satisfied by a biomedical product.

     

The Bifunctional Liposomes Constructed by Poly(2-ethyl-oxazoline)-cholesteryl Methyl Carbonate: an Effectual Approach to Enhance Liposomal Circulation Time,pH-Sensitivity and Endosomal Escape

Purpose

A novel bifunctional liposome with long-circulating and pH-sensitive properties was constructed using poly(2-ethyl-oxazoline)-cholesteryl methyl carbonate (PEtOz-CHMC) in this study.

Methods

PEtOz-CHMC was synthesized and characterized by TLC, IR and ¹H-NMR. The obtained PEtOz lipid was inserted into liposomes by the post-insertion method. Through a series of experiments, such as drug release, tumor cell uptake, cytotoxicity, calcium-induced aggregation, pharmacokinetic experiments, etc., the pH-sensitive and long-circulating properties of PEtOzylated liposomes was identified.

Results

PEtOz-CHMC modified liposomes (PEtOz-L) showed increased calcein release at low pH. Flow cytometric analysis results showed that the fusion and cellular uptake of PEtOz-L could be promoted significantly at pH 6.4 compared with those at pH 7.4. Confocal laser scanning microscope observations revealed that PEtOz-L could respond to low endosomal pH and directly released the fluorescent tracer into the cytoplasm. MTT assays in HeLa cells demonstrated that doxorubicin hydrochloride (DOX) loaded PEtOz-L exhibited stronger anti-tumor activity in a medium at pH 6.4 than in a medium pH 7.4. PEtOz-L remained stable when these liposomes were incubated in calcium chloride solution. The cumulative calcein release rate of PEtOz-L was significantly lower than that of CL when the liposomes were dialysed in PBS. The pharmacokinetic experiments of liposomes in rats showed that t _1/2 and AUC of PEtOz-L were 4.13 times and 4.71 times higher than those of CL.

Conclusions

PEtOzylated liposomes exhibits excellent long-circulating and pH-sensitive properties. Our results suggest that PEtOz is a promising biomaterial for the modification of liposome in drug delivery.     

Characterization of sequence-dependent synergy between ZD1839 ("Iressa") and oxaliplatin

ZD1839 ("Iressa"), a selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is currently undergoing preclinical and clinical evaluation in several solid tumors. The present study aimed to assess the effect of ZD1839 in combination with oxaliplatin in the colon cancer cell lines HT-29 and LoVo. For in vitro chemosensitivity testing, cells were treated with serial dilutions of each drug sequentially at a fixed ratio of doses that corresponded to 1/20, 1/10, 1/5, 1/2, 1, 1.5 and 2 times the individual IC(50) values. Oxaliplatin followed by ZD1839 produced a synergistic effect. In contrast, oxaliplatin following ZD1839 exhibited an additive effect at best. Mass spectrometry examination revealed that ZD1839 modestly enhanced cellular oxaliplatin accumulation and platinum-DNA (Pt-DNA) adducts (P>0.05). In additional studies, high-performance liquid chromatography revealed that oxaliplatin had no effect on ZD1839 accumulation. In contrast, ZD1839 markedly inhibited removal of Pt-DNA adducts (P<0.05). With oxaliplatin treatment (1 day) followed by ZD1839 (1 day), then incubation with drug-free medium (1 day), 90% of Pt-DNA adducts remained. Apoptosis examination revealed that oxaliplatin-induced apoptosis was prolonged by sequential oxaliplatin followed by ZD1839 treatment compared with oxaliplatin alone. Further experiments revealed that ZD1839 decreased cellular gamma-glutamyltransferase activity.CONCLUSIONS: The above observations provide a mechanistic explanation for the synergy of oxaliplatin followed by ZD1839, and suggest that this treatment combination warrants further preclinical and clinical investigation.     

Modulation of cellular redox state underlies antagonism between oxaliplatin and cetuximab in human colorectal cancer cell lines

Background and purpose:

Oxaliplatin is the first platinum-based compound effective in the treatment of colorectal cancer. Oxaliplatin combined with cetuximab for metastatic colorectal cancer is under evaluation. The preliminary results seem controversial, particularly for the use of cetuximab in K-Ras mutated patients. K-Ras mutation is known to affect redox homeostasis. Here we evaluated how the efficacy of oxaliplatin alone or combined with cetuximab varied according to the Ras mutation and redox status in a panel of colorectal tumour cell lines.

Experimental approach:

Viability was evaluated by methylthiazoletetrazolium assay, reactive oxygen species production by DCFDA and lucigenin on HT29-D4, Caco-2, SW480 and SW620 cell lines.

Key results:

Combination of oxaliplatin and cetuximab was less cytotoxic than oxaliplatin alone in colorectal cells harbouring wild-type Ras and membrane expression of receptors for epidermal growth factor receptor (EGFR), such as HT29-D4 and Caco-2 cells. In contrast, cetuximab did not affect oxaliplatin efficiency in cells harbouring K-Ras^V12 mutation, irrespective of membrane EGFR expression (SW620 and SW480 cells). Transfection of HT29-D4 with K-Ras^V12 decreased oxaliplatin IC₅₀ and impaired cetuximab sensitivity, without affecting expression of membrane EGFR compared with HT29-D4 control. Oxaliplatin efficacy relies on endogenous production of H₂O₂. Cetuximab inhibits H₂O₂ production inhibiting the EGFR/Nox1 NADPH oxidase pathway. Oxaliplatin efficacy was impaired by short hairpin RNA for Nox1 and by catalase (H₂O₂ scavenger).

Conclusions and implications:

Cetuximab limited oxaliplatin efficiency by affecting the redox status of cancer cells through Nox1. Such combined therapy might be improved by controlling H₂O₂ elimination.     

Electrospun Rapamycin-Eluting Polyurethane Fibers for Vascular Grafts

Purpose

To develop rapamycin-eluting electrospun polyurethane (PU) vascular grafts that could effectively suppress local smooth muscle cell (SMC) proliferation.

Methods

Rapamycin (RM) was incorporated in PU fibers by blend electrospinning using three distinct blending methods. The drug release profiles and the bioavailability of RM-containing PU fibers in the form of fibrous mats and vascular grafts were evaluated up to 77 days in vitro.

Results

RM-contained PU fibers generated by the three distinct blending methods exhibited significantly different fiber diameters (200–500 nm) and distinct RM release kinetics. Young’s moduli of the electrospun fibrous mats increased with higher RM contents and decreased with larger fiber diameters. For all blending methods, RM release kinetics was characteristic of a Fickian diffusion for at least 77 days in vitro. RM-PU fibers generated via powder blending showed the highest encapsulation efficiency. The RM in grafts made of these fibers remained bioactive and was still able to inhibit smooth muscle cell proliferation after 77 days of continual in vitro release.

Conclusions

Electrospun RM-containing PU fibers can serve as effective drug carriers for the local suppression of SMC proliferation and could be used as RM-eluting scaffolds for vascular grafts.     

Magnetic Targeting of Novel Heparinized Iron Oxide Nanoparticles Evaluated in a 9L-glioma Mouse Model

Purpose

A novel PEGylated and heparinized magnetic iron oxide nano-platform (DNPH) was synthesized for simultaneous magnetic resonance imaging (MRI) and tumor targeting.

Methods

Starch-coated magnetic iron oxide nanoparticles (“D”) were crosslinked, aminated (DN) and then simultaneously PEGylated and heparinized with different feed ratios of PEG and heparin (DNPH1-4). DNPH products were characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and superconducting quantum interference device (SQUID). The magentic targeting of DNPH3, with appropriate amounts of conjugated PEG and heparin, in a mouse 9L-glioma subcutaneous tumor model was confirmed by magnetic resonance imaging (MRI)/electron spin resonance (ESR).

Results

DNPH3 showed long circulating properties in vivo (half-life >8 h, more than 60-fold longer than that of parent D) and low reticuloendothelial system (RES) recognition in liver and spleen. Protamine, a model cationic protein, was efficiently loaded onto DNPH3 with a maximum loading content of 26.4 μg/mg Fe. Magnetic capture of DNPH3 in tumor site with optimized conditions (I.D. of 12 mg/kg, targeting time of 45 min) was up to 29.42 μg Fe/g tissue (12.26% I.D./g tissue).

Conclusion

DNPH3 showed the potential to be used as a platform for cationic proteins for simultaneous tumor targeting and imaging.     

Lysozyme Release and Polymer Erosion Behavior of Injectable Implants Prepared from PLGA-PEG Block Copolymers and PLGA/PLGA-PEG Blends

Purpose

We evaluated the controlled release of lysozyme from various poly(D,L-lactic-co-glycolic acid) (PLGA) 50/50-polyethylene glycol (PEG) block copolymers relative to PLGA 50/50.

Methods

Lysozyme was encapsulated in cylindrical implants (0.8 mm diameter) by a solvent extrusion method. Release studies were conducted in phosphate buffered saline +0.02% Tween 80 (PBST) at 37°C. Lysozyme activity was measured by a fluorescence-based assay. Implant erosion was evaluated by kinetics of polymer molecular weight decline, water uptake, and mass loss.

Results

Lysozyme release from an AB15 di-block copolymer (15% 5 kDa PEG, PLGA 28 kDa) was very fast, whereas an AB10 di-block copolymer (with 10% 5 kDa PEG, PLGA 45 kDa) and ABA10 tri-block copolymer (with 10% 6 kDa PEG, PLGA 27 kDa) showed release profiles similar to PLGA. We achieved continuous lysozyme release for up to 4 weeks from AB10 and ABA10 by lysozyme co-encapsulation with the pore-forming and acid-neutralizing MgCO₃, and from AB15 by co-encapsulation of MgCO₃ and blending AB15 with PLGA. Lysozyme activity was mostly recovered during 4 weeks.

Conclusions

These block co-polymers may have utility either alone or as PLGA blends for the controlled release of proteins.     

The Antitumor Effect of a New Docetaxel-Loaded Microbubble Combined with Low-Frequency Ultrasound In Vitro: Preparation and Parameter Analysis

Purpose

To develop a novel docetaxel (DOC)-loaded lipid microbubbles (MBs) for achieving target therapy and overcoming the poor water-solubility drawback of DOC.

Methods

A novel DOC-loaded microbubble (DOC + MB) was prepared by lyophilization and the physicochemical properties including ultrasound contrast imaging of the liver were measured. The anti-tumor effect of the DOC + MBs combined with low-frequency ultrasound (LFUS; 0.8Hz, 2.56 W/cm², 50% cycle duty) on the DLD-1 cancer cell line was examined using an MTT assay.

Results

The physicochemical properties of the two tested formats of DOC + MBs (1.0 mg and 1.6 mg) was shown: concentration, (6.74?±?0.02)?×?10⁸ bubbles/mL and (8.27?±?0.15)?×?10⁸ bubbles/mL; mean size, 3.296?±?0.004 μm and 3.387?±?0.005 μm; pH value, 6.67?±?0.11 and 6.56?±?0.05; release rate, 3.41% and 12.50%; Zeta potential, ?37.95?±?7.84 mV and ?44.35?±?8.70 mV; and encapsulation efficiency, 54.9?±?6.21% and 46.3?±?5.69%, respectively. Compared with SonoVue, the DOC + MBs similarly enhanced the echo signal of the liver imaging. The anti-tumor effect of the DOC + MBs/LFUS group was significantly better than that of DOC alone and that of the normal MBs/LFUS groups.

Conclusions

The self-made DOC + MBs have potential as a new ultrasound contrast agent and drug-loaded microbubble, and can obviously enhance the antitumor effect of DOC under LFUS exposure.     

Riboflavin-Targeted Polymer Conjugates for Breast Tumor Delivery

Purpose

In breast cancer, a significant decrease in riboflavin (RF) serum levels and increase in RF carrier protein occurs, indicating a potential role of RF in disease progression. To evaluate RF’s ability to serve as a targeting agent, mitomycin C (MMC)-conjugated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized and targeted to the RF internalization pathway in human breast cancer cells.

Methods

Competitive uptake studies were used to determine specificity of RF-targeted conjugates, and an MTT assay established the IC₅₀ for the conjugates. Endocytic mechanisms were investigated by confocal microscopy.

Results

Studies revealed a high-affinity endocytic mechanism for RF-specific internalization of fluorescently-labeled conjugates in both MCF-7 and SKBR-3 cells, whereas folic acid-mediated endocytosis showed high specificity only in SKBR-3 cells. MMC internalization was significantly higher following nontargeted and RF-targeted MMC-conjugate administration compared to that of free MMC. Cytotoxic analysis illustrated potent IC₅₀ values for RF-targeted MMC conjugates similar to free MMC. Maximum nuclear accumulation of MMC resulted from lysosomal release from RF-targeted and nontargeted MMC-conjugates following 6 h incubations, unlike that of free MMC seen within 10 min.

Conclusion

Targeting polymer-MMC conjugates to the RF internalization pathway in breast cancer cells enabled an increase in MMC uptake and nuclear localization, resulting in potent cytotoxic activity.