Preparation of thermosensitive PNIPAm-based copolymer coated cytodex 3 microcarriers for efficient nonenzymatic cell harvesting during 3D culturing.

Enzymatic detachment of cells might damage important features and functions of cells and could affect subsequent cell-based applications. Therefore, nonenzymatic cell detachment using thermosensitive polymer matrix is necessary for maintaining cell quality after harvesting. In this study, we prepared thermosensitive PNIPAm-co-AAc-b-PS and PNIPAm-co-AAm-b-PS copolymers and low critical solution temperature (LCST) was tuned near to body temperature. Then, spin coated polymer films were prepared for cell adhesion and thermal-induced cell detachment. The alpha-step analysis and scanning electron microscope image of the films suggested that the thickness of the films depends on the molecular weight and concentration which ranged from 206 to 1330 nm for PNIPAm-co-AAc-b-PS and 97.5-497 nm for PNIPAm-co-AAm-b-PS. The contact angles of the films verified that the polymer surface was moderately hydrophilic at 37°C. Importantly, RAW264.7 cells were convincingly proliferated on the films to a confluent of >80% within 48 h and abled to detach by reducing the temperature. However, relatively more cells were grown on PNIPAm-co-AAm-b-PS (5%w/v) films and thermal-induced cell detachment was more abundant in this formulation. As a result, PNIPAm-co-AAm-b-PS (5%w/v) was further used to coat commercial cytodex 3 microcarriers for 3D cell culturing and interestingly enhanced cell detachment with preserved potential of recovery was observed at a temperature of below LCST. Thus, surface modification of microcarriers with thermosensitive PNIPAm-co-AAm-b-PS could be vital strategy for nonenzymatic cell detachment and to achieve adequate number of cells with maximum cell viability and functionality.

Sterically polymer-based liposomal complexes with dual-shell structure for enhancing the siRNA delivery.

The sterically polymer-based liposomal complexes (SPLexes) were formed by cationic polymeric liposomes and pH-sensitive diblock copolymer were studied for their capabilities in improving the stability with high efficiency of siRNA delivery. The SPLexes were formed a dual-shelled structure and uniform size distribution. The PEGylated outer shell could mitigate the phagocytosis and reduce the cytotoxicity. Moreover, the folated SPLexes improved 42.9× accumulation in vitro and 1.7× tumor uptake in vivo in contrast with nonfolated SPLexes. The protonated copolymer at low pH would improve the siRNA released into cytoplasm following SPLexes fusion with the endo/lysosome membrane and inhibited the protein expression to 75.6 ± 4.5% efficiently. Results of this study significantly contribute to efforts to develop lipoplexes based siRNA delivery systems.

Thermo/redox-responsive dissolvable gelatin-based microsphere for efficient cell harvesting during 3D cell culturing.

The use of microspheres for culturing adherent cells has been proven as an important method, allowing for obtaining adequate number of cells in limited space and volume of medium for the intended cell-based medical applications. However, the use of proteolytic enzymes for cell harvesting from the microsphere resulted in cell damage and loss of functionality. Therefore, in this study, we developed a novel redox/thermo-responsive dissolvable gelatin-based microsphere for successful cell proliferation and harvesting adequate high-quality cells using non-enzymatic cell detachment methods. Initially, a redox-induced dissolvable gelatin-based microsphere was successfully prepared using disulfide bonds as crosslinking agent, firmly stabilizing gelatin networks and forming a stable microsphere at physiological temperature. The optimized concentration of the crosslinking agent was 1.2 mM, which kept the microsphere stable for >120 h. The microsphere was then coated with PNIPAm-ALA copolymer via physical or chemical means, resulting in a positively charged thermosensitive surface. The positive charge derived from ALA in PNIPAm-ALA copolymer enhanced cell attachment, while the thermosensitive property of the copolymer enabled for temperature induced cell harvesting. When the temperature dropped below the LCST value of PNIPAm-ALA5 (33.4°C), the copolymer swelled and became more hydrophilic, allowing cells to be readily separated. The addition of reducing agents such as GSH, DTT and L-cysteine resulted in further cleavage of the disulfide bond in the microsphere and dissolution of the microsphere for complete cell detachment. Interestingly, cell attachment and proliferation were enhanced on microspheres coated with PNIPAm-ALA5 using diselenide as a crosslinking agent, and complete cell detachment was occurred within 15 min after adding 25 mM DTT followed by lowering the temperature (4°C). Therefore, the microsphere fabricated in this study was worthwhile for non-enzymatic cell detachment and has the potential to be used for cell expansion and harvesting adequate live cells of high quality and functionality for tissue engineering or cell therapy.

In vitro evaluation of hexagonal polymeric micelles in macrophage phagocytosis.

This paper develops a non-spherical polymeric micelle using an amphiphilic block copolymer and a porphyrin crystalline structure. The nanoscale polymer micelles were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM), revealing particle sizes of approximately 150 nm with a particular shape in the hexagonal lattice. The shape shows the selective uptake efficacy for the HeLa and macrophage cells, and inhibits phagocytosis against the macrophage.

Fabrication of electroactive polypyrrole-tungsten disulfide nanocomposite for enhanced in vivo drug release in mice skin.

The present study focused on the development of electric stimuli drug release carrier based on transition metal dicgalcogenides. First, tungsten disulfide (WS2) was exfoliated and functionalized using thiol chemistry with various thiol-terminated ligands such as thioglycolic acid (TGA), mercaptosuccinic acid (MSA), and 2-ethanethiol (2ET). The exfoliated WS2 underwent non-covalent coating with an electrically conductive polypyrrole (PPy) for functionalization, of which MSA-WS2-PPy achieved the highest 5-FU (anticancer drug) loading. An electrically-stimulated drug release experiment showed that TGA-WS2-PPy achieved a higher drug release (90%) than MSA-WS2-PPy (70%) and 2ET-WS2-Ppy (35%). The TGA-WS2-PPy exhibited swelling/recombination between PPY and MSA-WS2 substrate under electrical stimulation, resulting in the highest 5-FU release. From the MTT assay result, there was no significant toxicity observed for TGA-WS2-PPy-FU on HaCaT cells, indicating the biocompatibility of TGA-WS2-PPy-FU in the absence of electrical stimulation. However, HaCaT cells died when incubated with TGA-WS2-PPy-FU under electrical stimulation. Finally, Raman mapping studies for TGA-WS2-PPy drug release in the skin of nude mice demonstrated that the carrier penetrated deeper into the skin of the mice while other systems failed to exhibit significant effects under electrical stimulation. The present study offers a novel approach in developing a non-invasive electrically-stimulated drug release system based on WS2 and an externally-controlled delivery model.

Localized controlled release of bevacizumab and doxorubicin by thermo-sensitive hydrogel for normalization of tumor vasculature and to enhance the efficacy of chemotherapy.

In a malignant tumor, overexpression of pro-angiogenic factors like vascular endothelial growth factor (VEGF) provokes the production of pathologic vascular networks characterized by leaky, chaotically organized, immature, thin-walled, and ill-perfused. As a result, hostile tumor environment would be developed and profoundly hinders anti-cancer drug activities and fuels tumor progression. In this study, we develop a strategy of sequential sustain release of anti-angiogenic drug, Bevacizumab (BVZ), and anti-cancer drug, Doxorubicin (DOX), using poly (d, l-Lactide)- Poly (ethylene glycol) -Poly (d, l-Lactide) (PDLLA-PEG-PDLLA) hydrogel as a local delivery system. The release profiles of the drugs from the hydrogel were investigated in vitro which confirmed that relatively rapid release of BVZ (73.56 ±â€¯1.39%) followed by Dox (61.21 ±â€¯0.62%) at pH 6.5 for prolonged period. The in vitro cytotoxicity test revealed that the copolymer exhibited negligible cytotoxicity up to 2.5 mg ml-1 concentration on HaCaT and HeLa cells. Likeways, the in vitro degradation of the copolymer showed 41.63 ±â€¯2.62% and 73.25 ±â€¯4.36% weight loss within 6 weeks at pH 7.4 and 6.5, respectively. After a single intratumoral injection of the drug-encapsulated hydrogel on Hela xenograft nude, hydrogel co-loaded with BVZ and Dox displayed the highest tumor suppression efficacy for up to 36 days with no noticeable damage on vital organs. Therefore, localized co-delivery of anti-angiogenic drug and anti-cancer drug by hydrogel system may be a promising approach for enhanced chemotherapeutic efficacy in cancer treatment.

Enhancing the in vivo transdermal delivery of gold nanoparticles using poly(ethylene glycol) and its oleylamine conjugate.

In this study, we investigated the effect of (ethylene glycol) (PEG) and PEG-oleylamine (OAm) functionalization on the skin permeation property of gold nanoparticles (GNS) in vivo. Chemisorption of polymers onto GNS was verified by a red shift in the ultraviolet-visible spectrum as well as by a change in the nanoparticle surface charge. The physicochemical properties of pristine and functionalized nanoparticles were analyzed by ultraviolet-visible spectroscopy, zeta potential analyzer, and transmission electron microscopy. Transmission electron microscopy revealed that the interparticle distance between nanoparticles increased after GNS functionalization. Comparing the skin permeation profile of pristine and functionalized GNS, the follicular deposition of GNS increased twofold after PEG-OAm functionalization. Moreover, PEG- and PEG-OAm-functionalized nanoparticles were able to overcome the skin barrier and deposit in the deeper subcutaneous adipose tissue. These findings demonstrate the potential of PEG- and PEG-OAm-functionalized GNS in serving a multitude of applications in transdermal pharmaceuticals.

Formation of gold decorated porphyrin nanoparticles and evaluation of their photothermal and photodynamic activity.

A core-shell gold (Au) nanoparticle with improved photosensitization have been successfully fabricated using Au nanoparticles and 5,10,15,20 tetrakis pentafluorophenyl)-21H,23H-porphine (PF6) dye, forming a dyad through molecular self-assembly. Au nanoparticles were decorated on the shell and PF6 was placed in the core of the nanoparticles. Highly stable Au nanoparticles were achieved using PF6 with poly(N-vinylcaprolactam-co-N-vinylimidazole)-g-poly(D,L-lactide) graft copolymer hybridization. This was compared with hybridization using cetyltrimethylammonium bromide and polyethylene glycol-b-poly(D,L-lactide) for shell formation with PF6-Au. The resulting PF6-poly(N-vinylcaprolactam-co-N-vinylimidazole)-g-poly(D,L-lactide)-Au core-shell nanoparticle were utilized for photothermal and photodynamic activities. The spectroscopic analysis and zeta potential values of micelles revealed the presence of a thin Au layer coated on the PF6 nanoparticle surface, which generally enhanced the thermal stability of the gold nanoparticles and the photothermal effect of the shell. The core-shell PF6-Au nanoparticles were avidly taken up by cells and demonstrated cellular phototoxicity upon irradiation with 300W halogen lamps. The structural arrangement of PF6 dyes in the core-shell particles assures the effectiveness of singlet oxygen production. The study verifies that PF6 particles when companied with Au nanoparticles as PF6-Au have possible combinational applications in photodynamic and photothermal therapies for cancer cells because of their high production of singlet oxygen and heat.

The effect of PEG-5K grafting level and particle size on tumor accumulation and cellular uptake.

PEG-modified gold nanoparticles (PEG-modified GNs) with diameters of 40 nm and 70 nm were prepared to elucidate the effect of extent of PEG (M.W. 5000) grafting and particle size on tumor accumulation and cellular uptake. Flow cytometry reveals that cellular uptake is strongly related to the size of PEG-modified GNs, rather than the extent of PEG-5K grafting level. Cytotoxicity analysis based on the intracellular release of drugs showed that the 70 nm PEG-modified GNs have the higher cytotoxicity, beccause of their greater cellular uptake. Also, particle size, rather than PEG-5K grafting level affects tumor accumulation. However, PEG-5K grafting level significantly affects the accumulation of particles in the liver and spleen. This finding is important in determining the proper PEG-5K grafting level and particle size for designing nano-medicines.

Stimulated release of photosensitizers from graft and diblock micelles for photodynamic therapy.

To understand the effect of photosensitizer (PS) release from graft copolymer based micelles in photodynamic therapy (PDT), the two pH-sensitive and non-pH-sensitive graft copolymers, (poly(N-vinyly caprolactam)-g-poly(D,L-lactide) and poly(N-vinyly caprolactam-co-N-vinyl imidazole)-g-poly(D,L-lactide)), were synthesized and utilized for the encapsulation of protoporphyrin IX (PPIX) for in vitro and in vivo PDT studies. Photochemical internalization (PCI) was utilized to study the localization of pH- and non-pH-sensitive micelles uptake in the lysosome. After non-toxic light treatment, PPIX was found in the nucleus with pH-sensitive micelles, while PPIX was still localized in the lysosomal organism with the non-pH-sensitive micelles, as observed by confocal microscopy. Because the formation of singlet oxygen was observed for the block and graft micelles, dramatic differences in the cell viability could be ascribed to the damage occurring at the region where the PPIX was located. An in vivo study revealed that PPIX-loaded graft and diblock micelles presented prolonged blood circulation and enhanced tumor targeting ability. The PPIX released from g-CIM micelles on tumor site was further proved by ex vivo confocal image. In addition, non-pH-sensitive micelle-treated mice showed a better repression of tumor growth than PPIX-treated mice, which was likely due to the larger amount of PS localized in the tumor region still exhibiting therapeutic effects. Finally, effective PDT-induced inhibition of tumor growth was found in pH-sensitive micelle-treated mice. This work provides insight into PS-loaded graft and diblock micelles for the PDT of tumors.

Novel geometry type of nanocarriers mitigated the phagocytosis for drug delivery.

Target geometry for mitigating phagocytosis has garnered considerable attention recently in the drug delivery field. This study examined nanoparticles (NPs) with same volume but different shapes, namely, spherical NPs (SNPs) and hexagonal nanoprisms (HNPs), and analyzed their behaviors in vitro and in vivo. These NPs were constructed with a multifunctional block copolymer component, mPEG-b-P(HEMA-co-histidine-PLA). Geometry of SNPs and HNPs was controlled by adjusting copolymer properties and particle size was controlled by adjusting formulation parameters. Nanoparticle morphology had no effect in mitigating phagocytosis when NP size was 70 nm; however, morphology had a significant effect when NP size was 120 nm. The radioactivity-time curves for (99m)Tc-labeled NPs, fitted by the two-compartment pharmacokinetic model, show that the prolonged plasma distribution half-life of HNPs is indicative in the bloodstream. The in vitro and in vivo studies reveal that dual stealth characteristics, pegylation and hexagonal prism structure, of nanocarriers can be adopted in clinical application for safe and efficient delivery of cancer therapy.