Doping gadolinium versus lanthanum into hydroxyapatite particles for better biocompatibility in bone marrow stem cells.

Lanthanide ions (Ln3+) doped hydroxyapatite (HAP) particles are well established in biomedical areas. Although Ln elements are closely located in the periodic table and have plenty of similar characteristics, the minor differences in the effective ionic radii could cause alterations in the physicochemical and biological properties of HAP substitutes. The present study synthesized lanthanum-(La-) and gadolinium-(Gd-) doped HAP particles (La-HAP and Gd-HAP). And the effects of two types of particles on bone marrow stem cells (BMSCs) viability were also measured and compared in vitro. The results indicated that the Gd-HAP adsorbed more serum proteins from culture media and inhibited the new layer of apatite formation on its surface when comparing to La-HAP with a similar crystalline structure, particle size, and Zeta potential. These surface modifications can significantly reduce the cell adhesion of Gd-HAP, simultaneously decreasing the Gd-HAP particle uptake efficiency. Moreover, the cell viability of Gd-HAP remained higher than that of La-HAP in culture periods. We concluded that a slight variation in the effective ionic radii between Gd3+ and La3+ could alter the adsorption of serum proteins on the particles' surface, modulating subsequent cellular responses. The present work provides an interesting view that Gd-HAP is endowed with better cellular biocompatibility than La-HAP.

Synergistic benefits from a lignin-first biorefinery of poplar via coupling acesulfamate ionic liquid followed by mild alkaline extraction.

A novel mind-set, termed lignin-first biorefinery, is bewitching to synchronously boost lignin output for entirely lignocellulosic utilization. A lignin-first fractionation, using a food-additive derived ionic liquid (1-ethyl-3-methylimidazolium acesulfamate, emimAce) and mild alkaline pretreatments, was formed for the purposely isolating poplar lignin, whilst delivering a cellulose-rich substrate that can be easily available for enzymatic digestion. The emimAce-driven lignin, alkali-soluble lignin and hemicellulose, and accessible cellulose were sequentially gained. We introduce a lignin-first approach to extract the amorphous fractions, destroy the robust architecture, and reform cellulose-I to II, thereby advancing the cellulose bioconversion from 15.4 to 90.5%. A harvest of 70.7% lignin, 52.1% hemicellulose, and 330.1 mg/g glucose was fulfilled from raw poplar. A structural ''beginning-to-end'' analysis of lignin inferred that emimAce ions are expected to interact with lignin ß-aryl-ether due to their aromatic character. It was reasonable to derive benefits from lignin-first technique that can substantially augment the domain of biorefinering.

Single-walled carbon-nanohorns improve biocompatibility over nanotubes by triggering less protein-initiated pyroptosis and apoptosis in macrophages.

Single-walled carbon-nanohorns (SNH) exhibit huge application prospects. Notably, spherical SNH possess different morphology from conventional carbon nanotubes (CNT). However, there is a tremendous lack of studies on the nanotoxicity and mechanism of SNH, and their comparison with nanotubes. Here, the dissimilarity between SNH and CNT is found in many aspects including necrosis, pyroptosis, apoptosis, protein expression, hydrolases leakage, lysosome stress, membrane disturbance and the interaction with membrane proteins. The improved biocompatibility of SNH over four types of established CNT is clearly demonstrated in macrophages. Importantly, a key transmembrane protein, glycoprotein nonmetastatic melanoma protein B (GPNMB) is discovered to initiate the nanotoxicity. Compared to CNT, the weaker nano-GPNMB interaction in SNH group induces lower degree of cascade actions from nano/membrane interplay to final cell hypotoxicity. In conclusion, the geometry of single-construct unit, but not that of dispersive forms or intracellular levels of nanocarbons make the most difference.

A dual-mediated liposomal drug delivery system targeting the brain: rational construction, integrity evaluation across the blood-brain barrier, and the transporting mechanism to glioma cells.

As the global population ages, cancer rates increase worldwide, and degenerative diseases of the central nervous system (CNS), brain tumors, and inflammation threaten human health more frequently. We designed a dual-mediated (receptor-mediated and adsorption-mediated) liposome, named transferrin-cell penetrating peptide-sterically stabilized liposome (TF-CPP-SSL), to improve therapy for gliomas through combining molecular recognition of transferrin receptors (TF-Rs) on the blood-brain barrier (BBB) and glioma cells with the internalization and lysosomal escaping ability of CPP. Based on the systematic investigation of structure-activity relations on the cellular level, we constructed TF-CPP-SSL rationally by conjugating TF and CPP moieties to the liposomes via PEG3.4K and PEG2.0K, respectively, and found the optimum densities of TF and CPP were 1.8% and 4%, respectively. These liposomes had the highest targeting efficacy for brain microvascular endothelial cell and C6 cell uptake but avoided capture by normal cells. Fluorescence resonance energy transfer technology and coculture models of BBB and glioma C6 cells indicated that TF-CPP-SSL was transported across the BBB without drug leakage, liposome breakup, or cleavage of ligand. TF-CPP-SSL offered advantages for crossing the BBB and entering into glioma C6 cells. Real-time confocal viewing revealed that TF-CPP-SSL was entrapped in endosomes of glioma C6 cells and then escaped from lysosomes successfully to release the liposomal contents into the cytosol. Entrapped contents, such as doxorubicin, could then enter the nucleus to exert pharmacological effects.

A novel stealth liposomal topotecan with amlodipine: apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine thus leading to an enhanced antitumor activity in leukemia.

The objectives of the present study were to define whether amlodipine induces apoptosis and what mechanism is involved in the process in human resistant and non-resistant leukemia cells following co-administration of stealth liposomal topotecan with amlodipine, a novel antiresistant liposomes developed by our institution. In three leukemias, K562, HL-60, and multidrug resistant (MDR) HL-60, cytotoxicity of topotecan was potentiated by amlodipine, while topotecan alone was resistant to MDR HL-60 cells. In two selected K562 or MDR HL-60 cells, the apoptotic effects were increased by addition of amlodipine, showing a dose-dependent manner. The activities of caspase 3 and 7 (marked as caspase 3/7), and caspase 8 were significantly activated by topotecan with amlodipine co-treated as the stealth liposomes. The deletions of intracellular Ca2+ stores induced by amlodipine correlated with the activated activities of caspase 3/7, or 8, respectively. In xenograft model with MDR HL-60 in nude mice, antitumor activity of stealth liposomal topotecan with amlodipine was significantly enhanced as compared to that of stealth liposomal topotecan or topotecan alone. In conclusion, apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine through activation of caspase 8 and then 3/7 activities. The enhanced antitumor activities by stealth liposomal topotecan with amlodipine are mainly due to the potentiating apoptotic effect and reversing the resistance by amlodipine. Stealth liposomal encapsulation of anticancer agent with a modulator may provide a novel strategy for improving the chemotherapeutic effects.

Dynamic bio-adhesion of polymer nanoparticles on MDCK epithelial cells and its impact on bio-membranes, endocytosis and paracytosis.

Nowadays, concern about the use of nanotechnology for biomedical application is unprecedentedly increasing. In fact, nanosystems applied for various potential clinical uses always have to cross the primary biological barrier consisting of epithelial cells. However, little is really known currently in terms of the influence of the dynamic bio-adhesion of nanosystems on bio-membranes as well as on endocytosis and transcytosis. This was investigated here using polymer nanoparticles (PNs) and MDCK epithelial cells as the models. Firstly, the adhesion of PNs on cell membranes was found to be time-dependent with a shift of both location and dispersion pattern, from the lateral adhesion of mainly mono-dispersed PNs initially to the apical coverage of the PN aggregate later. Then, it was interesting to observe in this study that the dynamic bio-adhesion of PNs only affected their endocytosis but not their transcytosis. It was important to find that the endocytosis of PNs was not a constant process. A GM1 dependent CDE (caveolae dependent endocytosis) pathway was dominant in the preliminary stage, followed by the co-existence of a CME (clathrin-mediated endocytosis) pathway for the PN aggregate at a later stage, in accordance with the adhesion features of PNs, suggesting the modification of PN adhesion patterns on the endocytosis pathways. Next, the PN adhesion was noticed to affect the structure of cell junctions, via altering the extra- and intra-cellular calcium levels, leading to the enhanced paracellular transport of small molecules, but not favorably enough for the obviously increased passing of PNs themselves. Finally, FRAP and other techniques all demonstrated the obvious impact of PN adhesion on the membrane confirmation, independent of the adhesion location and time, which might lower the threshold for the internalization of PNs, even their aggregates. Generally, these findings confirm that the transport pathway mechanism of PNs through epithelial cells is rather dynamic, and is remarkably affected by the adhesion patterns of PNs on the cell membrane.

The modulation of tumor vessel permeability by thalidomide and its impacts on different types of targeted drug delivery systems in a sarcoma mouse model.

The transport of nanocarriers is supposed to be based on EPR effect which is affected by diverse factors, so the modulation of EPR effect seems very significant for nanocarriers including targeted drug delivery systems (TDDSs). Besides, it is extremely unclear how the EPR effect impacts the fate of different types of TDDSs. To make the most advantage of EPR effect for TDDSs, it is definitely necessary to clarify these key issues. Here, we construct and characterize various TDDSs, including sterically-stabilized liposomes (SSL), RGD functionalized SSL (RGD-SSL) and novel 7PEP functionalized SSL (7PEP-SSL), loaded with doxorubicin (DOX), DIR or DID. Here, we modulate the permeability of tumor vessels by thalidomide (THD) in a sarcoma-bearing EPR mouse model via monitoring endogenous deoxygenated hemoglobin in circulation, and then we confirm the effect of THD on tumor vessel permeability by vessel density, vessel maturity, VEGF expression and so on. Importantly, we investigate and find the impacts of EPR effect on the antitumor efficacy, in vivo distribution and intratumoral microdistribution of the three TDDSs. Interestingly, the EPR effects affect different TDDSs differently. The elevated EPR effect enhances the tumor accumulation of SSL and RGD-SSL but fails to increase their efficacy. The RGD-SSL exhibits the best efficacy with the least fluctuation, demonstrating the advantage of angiogenesis targeted systems. 7PEP-SSL seems the biggest beneficiary of EPR effect, suggesting the significance of EPR modulation for cells targeted systems. Generally, this study demonstrates the feasibility of modulating EPR effect bidirectionally by THD as well as the impacts of EPR effect on different type of testing TDDSs based on this animal model. It certainly provides novel insight into the design and potential use of TDDSs.

Comprehensively priming the tumor microenvironment by cancer-associated fibroblast-targeted liposomes for combined therapy with cancer cell-targeted chemotherapeutic drug delivery system.

Cancer-associated fibroblasts (CAFs) not only support tumorigenesis and tumor metastasis by reciprocal cellular cross-talk with cancer cells, but also remodel the extracellular matrix (ECM) and architecture of tumor microenvironment. This leads to poor tumor penetration of traditional chemotherapeutic nanomedicines and resulting drug resistance. In this study, we use a novel tumor stroma-targeted nanovehicle (FH-SSL-Nav) to specifically eradicate CAFs, promote tumor penetration of nanomedicines and cut off the stroma's support to cancer cells. FH-SSL-Nav exhibited excellent and comprehensive tumor microenvironment modulation including downregulation ECM deposition, decreasing interstitial fluid pressure (IFP) and facilitating blood perfusion. As a result, more chemotherapeutic drug delivery systems penetrated deep into tumor spheroids in vitro and tumor tissues in vivo. Furthermore, chemotherapeutic drug resistance induced by microenvironment was partly reversed by FH-SSL-Nav. In a human Hep G2 xenograft nude mouse model, FH-SSL-Nav greatly improved the tumor suppression of cancer cell-targeted liposomal doxorubicin (7pep-SSL-DOX) with low dose and low toxicity. Since Nav and DOX exhibited no synergy against Hep G2 cells, it was clear that the improved antitumor efficacy was basically due to the comprehensive tumor microenvironment priming by FH-SSL-Nav.

Dextran-coated fluorapatite crystals doped with Yb3+/Ho3+ for labeling and tracking chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro and in vivo.

Upconversion fluorescent nanoparticles are becoming more widely used as imaging contrast agents, owing to their high resolution and penetration depth, and avoidance of tissue auto-fluorescence and photodamage to cells. Here, we synthesized upconversion fluorescent crystals from rare-earth Yb3+ and Ho3+ co-doped fluorapatite (FA:Yb3+/Ho3+) suitable for long-term tracking and monitoring cartilage development (chondrogenesis) in bone marrow mesenchymal stem cells (BMSCs) in vitro and in vivo. We initially determined the structure, morphology and luminescence of the products using X-ray powder diffraction, transmission electron microscopy and two-photon confocal microscopy. When excited at 980 nm, FA:Yb3+/Ho3+ crystals exhibited distinct upconversion fluorescence peaks at 543 nm and 654 nm. We then conjugated FA:Yb3+/Ho3+ crystals with dextran to enhance hydrophilicity, biocompatibility and cell penetration. Next, we employed the dextran-coated FA:Yb3+/Ho3+ crystals in labeling and tracking chondrogenic differentiation processes in BMSCs stably expressing green fluorescent protein (BMSCsGFP) in vitro and in vivo. Labeled BMSCsGFP were shown to reproducibly exhibit chondrogenic differentiation potential in RT-PCR analysis, histological assessment and immunohistochemistry. We observed continuous luminescence from the FA:Yb3+/Ho3+ upconversion crystals at 4 weeks and 12 weeks post transplantation in BMSCsGFP, while GFP fluorescence in both control and crystal-treated groups significantly decreased at 12 weeks after BMSCsGFP transplantation. We therefore demonstrate the high biocompatibility and stability of FA:Yb3+/Ho3+ crystals in tracking and monitoring BMSCs chondrogenesis in vitro and in vivo, highlighting their excellent cell labeling potential in cartilage tissue engineering.

Dual targeting for metastatic breast cancer and tumor neovasculature by EphA2-mediated nanocarriers.

EphA2 is a transmembrane receptor tyrosine kinase that is highly expressed on both tumor neovasculature and some kinds of tumor cells. Here, a homing peptide with a sequence of YSAYPDSVPMMSK (YSA) that binds specifically with EphA2 was utilized to modify the stealth liposomes (YSA-LP). With a particle size of about 85 nm, this functionalized nanocarrier was loaded with fluorescent probe or doxorubicin (DOX) and investigated in vitro and in vivo. In the cellular endocytosis studies in vitro, coumarin-6 loaded YSA-LP exhibited significant specificity to both EphA2-overexpressing tumor cells (MDA-MB-231) and human umbilical vein endothelial cells (HUVEC) via a YSA mediated interaction. In a MDA-MB-231 xenograft tumor mouse model, DiR-loaded YSA-LP showed more lasting accumulation in tumor tissue by small animal imaging compared to unmodified liposomes (LP). Further, YSA-LP greatly facilitated the efficacy of DOX loaded against both tumor cells and tumor angiogenesis in the same mouse model, evidenced by inhibiting tumor growth, metastasis and CD31 expression as well as inducing cancer cell apoptosis. Additionally, YSA-LP (DOX) showed relatively low systemic and cardiac toxicity compared with control groups. In conclusion, YSA might be a promising targeting motif for EphA2-overexpressing tumor cells and tumor neovasculature, which could be used to mediate drug delivery for chemotherapy agents.

PSMA-mediated endosome escape-accelerating polymeric micelles for targeted therapy of prostate cancer and the real time tracing of their intracellular trafficking.

The cytotoxicity of chemotherapeutic agents to healthy organs and drug resistance of tumor cells are believed to be the main obstacles to the successful cancer chemotherapy in the clinic. To ensure that anticancer drugs could be delivered to the tumor region, are quickly released from carriers in tumor cells and rapidly escape from endo/lysosomes, YPSMA-1-modified pH-sensitive polymeric micelles, which would be advantageous in recognizing the prostate specific membrane antigen (PSMA), were designed and fabricated for targeted delivery of paclitaxel to tumors based on the pH-sensitive diblock copolymer poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) (PEOz-PLA) and YPSMA-1-PEOz-PLA for treating prostate cancer. HOOC-PEOz-PLA with a critical micelle concentration of 5.0 mg L(-1) was synthesized and characterized by (1)H NMR and gel permeation chromatography. The prepared YPSMA-1-modified micelles, about 30 nm in diameter, exhibited a rapid release behavior at endo/lysosome pH and a favorable ability of fast endo/lysosome escape as observed by confocal microscopy. More importantly, we evidenced for the first time that both endosome and lysosome escape existed for pH-sensitive micelles via real time tracing using confocal microscopy, and the real time endo/lysosome escape process was also presented. The YPSMA-1-modified micelles were very effective in enhancing the cytotoxicity of paclitaxel by increasing the cellular uptake in PSMA-positive 22Rv1 cells, which was verified the correlation with PSMA expression in tumor cells by flow cytometric analysis and confocal microscopy. Moreover, the active targeting and pH-sensitivity endowed YPSMA-1-modified micelles with a higher antitumor efficacy and negligible systemic toxicity in 22Rv1 xenograft-bearing nude mice compared with unmodified micelles and Taxol®. These results suggested that the application of combining YPSMA-1 modification with pH-sensitivity to polymeric micelles may be one approach in the efficient delivery of anticancer drugs for treating PSMA-positive prostate cancers.

The co-delivery of a low-dose P-glycoprotein inhibitor with doxorubicin sterically stabilized liposomes against breast cancer with low P-glycoprotein expression.

INTRODUCTION: P-glycoprotein (P-gp) inhibitors are usually used to treat tumors that overexpress P-gps. However, most common types of breast cancers, such as Luminal A, are low-P-gp expressing, at least during the initial phases of treatment. Therefore, it would be interesting to know if P-gp inhibitors are still useful in treating low-P-gp-expressing tumors. METHODS: In the study reported here, the human breast-cancer cell line MCF-7, chosen as a model of Luminal A, was found to be low-P-gp expressing. We designed a novel doxorubicin (DOX) sterically stabilized liposome system co-loaded with the low-dose P-gp inhibitor cyclosporine A (CsA) (DOX/CsA/SSL). RESULTS: The co-delivery system showed good size uniformity, high encapsulation efficiency, and a desirable release profile. The cell-uptake and cytotoxicity studies demonstrated that CsA could significantly enhance the intracellular accumulation and toxicity of free DOX and the liposomal DOX in MCF-7 cells. The confocal microscopy and in vivo imaging study confirmed the intracellular and in vivo targeting effect of DOX/CsA/SSL, respectively. Finally, the in vivo study proved that DOX/CsA/SSL could achieve significantly better antitumor effect against MCF-7 tumor than controls, without inducing obvious systemic toxicity. CONCLUSION: This study demonstrated that the co-delivery of a low-dose P-gp inhibitor and liposomal DOX could improve the therapy of low-P-gp-expressing cancer, which is of significance in clinical tumor therapy.

A functional biphasic biomaterial homing mesenchymal stem cells for in vivo cartilage regeneration.

Cartilage regeneration after trauma is still a great challenge for clinicians and researchers due to many reasons, such as joint load-bearing, synovial movement and the paucity of endogenous repair cells. To overcome these limitations, we constructed a functional biomaterial using a biphasic scaffold platform and a bone-derived mesenchymal stem cells (BMSCs)-specific affinity peptide. The biphasic scaffold platform retains more cells homogeneously within the sol-gel transition of chitosan and provides sufficient solid matrix strength. This biphasic scaffold platform is functionalized with an affinity peptide targeting a cell source of interest, BMSCs. The presence of conjugated peptide gives this system a biological functionality towards BMSC-specific homing both in vitro and in vivo. The functional biomaterial can stimulate stem cell proliferation and chondrogenic differentiation during in vitro culture. Six months after in vivo implantation, compared with routine surgery or control scaffolds, the functional biomaterials induced superior cartilage repair without complications, as indicated by histological observations, magnetic resonance imaging and biomechanical properties. Beyond cartilage repair, this functional biphasic scaffold may provide a biomaterial framework for one-step tissue engineering strategy by homing endogenous cells to stimulate tissue regeneration.

A specific peptide ligand-modified lipid nanoparticle carrier for the inhibition of tumor metastasis growth.

Tumor metastasis accounts for 90% of cancer-associated deaths and is almost inaccessible by chemotherapy, surgical operation or radiotherapy. Here, a tumor metastasis-specific nanocarrier system has been constructed by modification of stealth lipid nanoparticles with a specific peptide ligand. Highly metastatic breast cancer MDA-MB-231 that stably expressed luciferase (MDA-MB-231/Luc) was used as tumor cell model. The nanocarrier was very specific for highly metastatic cancer cells in vitro and could specifically target to cancer metastases foci following systemic administration in vivo by both fluorescence imaging and bioluminescence imaging compared to a passive-targeted system. It greatly facilitated the efficacy of doxorubicin loaded in inhibiting tumor metastasis growth and prolonging the survival time of mice. Importantly, this system was also found to prevent the initiation and progression of tumor metastasis. The tumor metastasis-targeted nanocarriers hold great potential in the treatment of cancer metastasis foci and even for the prevention of tumor metastasis. This study may also provide new strategy in the development of nanomedicine for diagnosis and therapy of tumor metastasis.

Transferrin receptor specific nanocarriers conjugated with functional 7peptide for oral drug delivery.

In an attempt to increase the interaction of a nanocarrier system with gastrointestinal epithelial cells, a transferrin receptor (TfR) specific 7peptide was conjugated to PEG-b-PCL copolymer and the functional nanocarriers were constructed and characterized. The endocytosis, intracellular trafficking and transcytosis of such nanocarriers loaded with coumarin 6 (7pep-M-C6) in a human colon carcinoma cell line (Caco-2) were investigated, followed by the in vivo intestine distribution study. The real-time imaging of live cell, three dimensional reconstruction of confocal image, quantitative colocalization analysis and other techniques were applied. First, the TfR expression was confirmed in Caco-2. Then, 7pep-M-C6 exhibited higher intracellular uptake compared with unmodified nanocarriers. In a live cell study, 7pep-M-C6 demonstrated faster uptake kinetics especially in the surface of cells. Together with a competition study using TfR antibody, it was proved that the increased cellular uptake was due to a receptor-mediated mechanism. Besides the unspecific endocytosis pathway, 7pep-M-C6 was found to enter the cells through a specific clathrin-mediated mechanism, related to the expression of TfR on Caco-2 cells. Possibly for this reason, 7pep-M-C6 tended to colocalize more with late endosomes and lysosomes than the control micelles. Also for the same mechanism, the increased transport of 7pep-M-C6 across Caco-2 monolayer was found, through a transcellular but not a paracellular pathway, while an increased in vivo intestinal distribution of 7pep-M-C6 was observed. In conclusion, the functional nanocarriers could specifically interact with gastrointestinal endothelial cells, increase their transport and alter their pathway as a result.

The reduction of tumor interstitial fluid pressure by liposomal imatinib and its effect on combination therapy with liposomal doxorubicin.

Interstitial fluid pressure (IFP) in tumor is much higher than that in normal tissue and it constitutes a great obstacle for the delivery of chemodrugs, which makes it a potential target for cancer therapy. In this study, imatinib, a molecular targeting drug, was loaded in sterically stabilized liposomes (SSL-IMA) to reduce the tumor IFP, in an attempt to deliver more liposomal doxorubicin (SSL-DOX) into tumor tissue. In a mouse B16 melanoma model, intravenous injection of 20 mg/kg SSL-IMA achieved the most reduction of tumor IFP and the effect lasted for at least 50 h with the least hematotoxicity. However, intragastric administration of 100 mg/kg free IMA did not decrease the tumor IFP significantly. Mechanisms of the reduction of tumor IFP by SSL-IMA were proved to be the inhibition of PDGF receptor beta, the inhibition of tumor fibroblasts as well as the anti-angiogenesis effect of SSL-IMA. Then it was demonstrated by in vivo imaging that the decrease of tumor IFP by SSL-IMA led to a more and longer intratumoral distribution of the lipid vehicles. The improved delivery was proved again in the anti-tumor study. The combination of SSL-IMA and SSL-DOX inhibited tumor growth and induced apoptosis of tumor cells the most, at a low dose in which neither SSL-DOX nor SSL-IMA showed obvious anti-tumor efficacy. Since no synergy against B16 cells was found between SSL-IMA and SSL-DOX, it was clear that the improved combinational therapy was basically due to the decrease of tumor IFP by SSL-IMA. In conclusion, reducing tumor IFP by SSL-IMA seems to be a promising strategy to potentiate chemotherapies.

The transport pathways of polymer nanoparticles in MDCK epithelial cells.

Epithelial cell membranes as the typical biological barrier constitute the prime obstacle for the transport of therapeutic agents including nanomedicines. The previous studies on the interaction between nanomedicines and cells are mostly emphasized on cellular uptake and intracellular trafficking, but seldom on epithelial cells, although more and more oral nanomedicines are available now. In an attempt to clarify the transport pathways of nanomedicines in epithelial cells, the different molecular mechanisms among endocytosis, exocytosis and transcytosis processes were carefully studied and compared here using a kind of polymer nanoparticles (PNs) and MDCK epithelial cells as models. As the result, their similarity and difference were demonstrated. The similarities among all the three processes included the mediation of lipid rafts, the involvement of some protein kinases such as protein tyrosine kinase (PTK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), and the existence of multiple pathways. However, the difference among these processes was very significant, including different pathways, and especially the disparate effects of lipid rafts and protein kinases for different processes. The endocytosis involved both lipid raft and clathrin mechanisms but no macropinocytosis, via the invagination of membrane but no pore formation, the exocytosis contained ER/Golgi and Golgi/PM pathways, and transcytosis included AEE/CE/BSE and Golgi/BSE pathways. The roles of lipid rafts on endocytosis were positive but that on exocytosis and transcytosis was negative. The impacts of PTK and PKC on endocytosis were positive, while the influences of PTK, PKC and P13K on AEE/CE/BSE, as well as PTK and P13K on Golgi/BSE transcytosis pathways were negative. Moreover, the discrepancy between inward and outward transport of PNs elucidated an interesting fact that the endocytosis was rather easy and outward transport including exocytosis and transcytosis was rather difficult. Finally, it was indicated by comparison with previous reports that the molecular mechanisms between PNs and macromolecules such as proteins were also dissimilar.

The transport mechanisms of polymer nanoparticles in Caco-2 epithelial cells.

As the primary physiological barrier, intestinal epithelial cells regulate the transportation of oral therapeutic agents including nanomedicines which significantly improves the bioavailability of many drugs. However, currently there seems in the lack of comprehensive understanding on nanoparticle transport in intestinal epithelial cells as well as the mechanisms related. So, in an attempt to illustrate the profile of nanoparticle transport in intestinal epithelial cells, Caco-2 cells and polymer nanoparticles (PNs) were used as the models to explore the whole transport process including endocytosis, intracellular trafficking, exocytosis and transcytosis. Via various techniques, the transport pathways of PNs in Caco-2 cells and their mechanisms were clarified. Firstly, the transport was characterized by its non-specificity. The co-mediation of clathrin, lipid raft/caveolae and macropinocytosis as well as the co-involvement of different proteins like actins, protein tyrosine kinase (PTK) and cyclooxygenase (COX) were found in the endocytosis of PNs. The endocytosed PNs could transport to apical early endosome (AEE) and then from AEE to lysososmes via AEE/late endosome (LE)/lysosome pathway, as well as to recycling endosome compartment (REC) or endoplasmic reticulum (ER) through AEE/REC and AEE/ER pathways, respectively. Both ER/Golgi and Golgi/REC/plasma membrane (PM) pathways were involved in the exocytosis of PNs. The transcytosis of PNs across the cell monolayer was very low with a ratio less than 0.5%, due to complicated reasons. Secondly, the transport was evidenced by its partial energy-dependency. Beside the energy-dependent transport mediated by some proteins, quantitative study demonstrated the obvious internalization as well as surface binding of PNs at both 37 °C and 4 °C, but significantly higher at 37 °C. Interestingly, the consistency between surface binding and internalization at each temperature was found, suggesting that cell binding was the precondition and key step for the following endocytosis. The involvement of both energy dependent and independent mechanism was also observed in the exocytosis and transcytosis process of PNs. Finally, there were opposite mechanisms found between the exocytosis and endocytosis of PNs, including the regulation role of lipid raft/caveolae, COX and Golgi complex, which also contributed to the fact of "easy entry and hard across" for PNs. Overall, this study depicts a clear picture of nanoparticle transport in Caco-2 epithelial cells characterized by non-specificity, partial energy-dependency and low transcytosis.

The effect of hydrophilic and hydrophobic structure of amphiphilic polymeric micelles on their transport in epithelial MDCK cells.

The interaction of nanocarriers with cells including their transcellular behavior is vital not only for a drug delivery system, but also for the safety of nanomaterials. In an attempt to clarify how the structures of polymers impact the transport mechanisms of their nanocarriers in epithelial cells, three amphiphilic polymers (PEEP-PCL, PEG-PCL and PEG-DSPE) with different hydrophilic or hydrophobic blocks were synthesized or chosen to form different micelle systems here. The endocytosis, exocytosis, intracellular colocalization, paracellular permeability and transcytosis of these micelle systems were compared using Förster resonance energy transfer analysis, real-time confocal images, colocalization assay, transepithelial electrical resistance study, and so on. All micelle systems were found intact during the studies with cells. The endocytosis and exocytosis studies with undifferentiated MDCK cells and the transcytosis study with differentiated MDCK monolayers all indicated the fact that PEG-DSPE micelles achieved the most and fastest transport, followed by PEG-PCL and PEEP-PCL in order. These might be because DSPE has higher hydrophobicity than PCL while PEG has lower hydrophilicity than PEEP. Different in hydrophilic or hydrophobic structures, all kinds of micelles demonstrated similar pathways during endocytosis and exocytosis, both caveolae- and clathrin-mediated but with difference in degree. The colocalization studies revealed different behaviors in intracellular trafficking among the three polymer micelles, suggesting the decisive role of hydrophilic shells on this process. Finally, all micelle systems did not impact the paracellular permeability of test cell monolayer. In conclusion, the hydrophilic and hydrophobic structures of test micelles could influence their transport ability, intracellular trafficking and the transport level under each pathway in MDCK cells.

Novel albendazole-chitosan nanoparticles for intestinal absorption enhancement and hepatic targeting improvement in rats.

To improve the treatment of helminthiasis, filariasis, and colorectal cancer, albendazole-associated chitosan nanoparticles (ABZ-CS-NPs) were prepared using the emulsion crosslinking volatile technique with contained sodium tripolyphosphate as the crosslinking agent and Poloxamer 188 as the auxiliary solvent. The structural characteristics of the NPs were determined using X-ray diffraction to analyze the interaction between CS and the drug. The NPs were then evaluated in terms of their physicochemical characteristics, drug release behavior, in vivo pharmacokinetic parameters, and biodistribution in animal studies. ABZ-loaded NPs with a uniformly spherical particle sizes (157.8 ± 2.82 nm) showed efficient drug loading, encapsulated efficiency, and high physical stability. The drug release from ABZ-CS-NPs was extended over several periods. Kinetic models were then fitted to determine the release mechanisms. ABZ and its metabolite albendazole sulfoxide (ABZSX) were analyzed in rats with mebendazole as the internal standard using reversed-phase high-performance liquid chromatography. Compared with the ABZ suspension groups, the relative bioavailability values of ABZ and ABZSX were 146.05 and 222.15%, respectively. In addition, the plasma concentration versus time curve is consistent with that of the two compartment models in the plasma concentration versus time curve. The results indicate that the ABZ-loaded NPs are promising novel ABZ candidates for passive diffusion in the treatment of hydatid cysts in the liver via oral administration.