Multilayer Coating of Tetrandrine-loaded PLGA Nanoparticles: Effect of Surface Charges on Cellular Uptake Rate and Drug Release Profile

The effect of surface charges on the cellular uptake rate and drug release profile of tetrandrine-loaded poly（lactic-co-glycolic acid）（PLGA） nanoparticles（TPNs） was studied. Stabilizer-free nanoprecipitation method was used in this study for the synthesis of TPNs. A typical layer-by-layer approach was applied for multi-coating particles＇ surface with use of poly（styrene sulfonate） sodium salt（PSS） as anionic layer and poly（allylamine hydrochloride）（PAH） as cationic layer. The modified TPNs were characterized by different physicochemical techniques such as Zeta sizer, scanning electron microscopy and transmission electron microscopy. The drug loading efficiency, release profile and cellular uptake rate were evaluated by high performance liquid chromatography and confocal laser scanning microscopy, respectively. The resultant PSS/PAH/PSS/PAH/TPNs（4 layers） exhibited spherical-shaped morphology with the average size of 160.3±5.165 nm and zeta potential of –57.8 m V. The encapsulation efficiency and drug loading efficiency were 57.88% and 1.73%, respectively. Multi-layer coating of polymeric materials with different charges on particles＇ surface could dramatically influence the drug release profile of TPNs（4 layers vs. 3 layers）. In addition, variable layers of surface coating could also greatly affect the cellular uptake rate of TPNs in A549 cells within 8 h. Overall, by coating particles＇ surface with those different charged polymers, precise control of drug release as well as cellular uptake rate can be achieved simultaneously. Thus, this approach provides a new strategy for controllable drug delivery.

Preparation and cellular uptake behaviors of uniform fiber-like micelles with length controllability and high colloidal stability in aqueous media

Fragmentation/disassembly of fiber-like micelles generated by living crystalline-driven self-assembly(CDSA)is usually encountered in aqueous media,which hinders the applications of micelles.Herein,we report the generation of uniform fiber-like micelles consisting of a𝜋Л-conjugated oligo(p-phenylenevinylene)core and a crosslinking silica shell with grafted poly(ethylene glycol)(PEG)chains by the combination of living CDSA,silica chemistry and surface grafting-onto strategy.Owing to the presence of crosslinking silica shell and the outmost PEG chains,the resulting micelles exhibit excellent dispersity and colloidal stability in PBS buffer,BSA aqueous solution and upon heating at 80℃ for 2 h without micellar fragmentation/disassembly.The micelles also show negligible cytotoxicity toward both HeLa cervical cancer and HEK239T human embryonic kidney cell lines.Interestingly,micelles with Ln of 156 nm show the“stealth”property with no significant uptake by HeLa cells,whereas some certain amounts of micelles with Ln of 535 nm can penetrate into HeLa cells,showing length-dependent cellular uptake behaviors.These results provide a route to prepare uniform,colloidally stable fiber-like nanostructures with tunable length and functions derived for biomedical applications.

Construction of folate-conjugated epirubicin liposomes for enhancing the cellular uptake and the co-localization with nuclei of invasive breast cancer cells

Drug resistance of anthracycline in the invasive cancer is associated with the lowered cellular drug uptake and diminished co-localization of drug with nuclei. In the present study, we aimed to construct the folate-conjugated epirubicin liposomes by incorporating a synthesized folate-lipid derivative; and to assess the effects on cellular drug uptake, co-localization of drug with nuclei and efficacy in treatment of invasive breast cancer cells. The studies were performed on invasive human breast cancer cells. The folate-PEG2ooo-DSPE conjugate was synthesized, and the constructed folate-conjugated epirubicin liposomes were approximately 1 O0 nm in size. The in vitro studies demonstrated that the folate-conjugated epirubicin liposomes had the strongest cellular drug uptake and co-localization with nuclei of the invasive breast cancer cells. Besides, the liposomes displayed the most significant efficacy in killing the invasive cancer cells, in preventing their invasive potential, and in penetrating ability into breast cancer spheroid as well. In conclusion, the constructed folate-conjugated epirubicin liposomes were able to enhance the efficacy in treatment of invasive breast cancer by improving the cellular drug uptake and increasing the co-localization with nuclei, hence offering a new strategy for potentially eradicating the invasive breast cancer cells.

Design of nanocarriers for efficient cellular uptake and endosomal release of small molecule and nucleic acid drugs： learning from virus

There are many challenges in developing efficient and target specific delivery systems of small molecule and nucleic acid drugs. Cell membrane presents one of the major barriers for the penetration of hydrophilic macromolecules across the plasma membrane. Nanocar- riers have been designed to enhance their cellular uptake via endocytosis but following their cellular uptake, endosomal escape is the rate limiting step which restricts the value associated with the enhanced uptake by nanocarriers. Viruses are an excellent model for efficient cytosolic delivery by nanocarriers. Viruses exploit intra- cellular cues to release the genome to cytosol. In this review, we first discuss different endocytic uptake path- ways and endosomal escape mechanisms. We then summarize the existing tools for studying the intracellular trafficking of nanocarriers. Finally, we highlight the important design elements of recent virus-based nanocar- tiers for efficient cellular uptake and endosomal escape.

EFFECT OF SURFACE CHARGE OF POLYMERIC MICELLES ON IN VITRO CELLULAR UPTAKE

This work focuses on the interaction between polymeric micelles with different charged surfaces and cancer cells in order to study the influence of surface charge on the in vitro cellular uptake efficiency. The amphiphilic diblock copolymers poly（e-caprolaetone）-b-poly（ethylene oxide） （PCL-b-PEO） with different functional groups at the end of hydrophilic block were synthesized. The functional groups endue the micelles with different charges on the surfaces. The cellular uptake of micelles to T-24 cells （human bladder tumor cells）, HepG2 cells （human liver hepatocellular carcinoma cell line） and Hela cells （human epithelial cervical cancer cells） was studied by means of flow cytometer and confbcal laser scanning microscopy. The results indicate that the surface charges showed great influence on zeta potential of micelles at different pH values. The in vitro cellular uptake efficiency of micelles with different charged surfaces demonstrated different cellular uptake patterns to three kinds of cancer cells.

Gold Nanoparticle Enantiomers and Their Chiral-Morphology Dependence of Cellular Uptake

Chiral molecules are widely prevalent in nature and biological systems,and artificial chiral nanoparticles have drawn enormous interest owing to their unique optical and physical properties.However,nanoparticles with chiral morphologies and their potential role in biology have been rarely explored.Herein,we report a seed-mediated synthesis of enantiomorphic Au nanooctopods(NOPs)and their chiralmorphology dependence of cellular uptake.With a high yield(∼80%),the chiral NOPs possess eight uniform arms that bend from<111>to<100>directions,like a propeller structure.The chiral NOPs synthesized with L-or D-glutathione(GSH)have opposite handedness,resulting in opposite circular dichroism signals,which is consistent with finite-difference time-domain simulations.D-GSH NOPs demonstrate greater than 30%(ca.15%)enhanced cellular uptake in GL261 and bEnd.3 cells compared with L-GSH NOPs(racemic NOPs).Moreover,D-GSH NOPs modified with poly(ethylene glycol)or L-GSH are also preferred by the cells,proving the chiral-morphology dependence of cellular uptake.Our study develops the exploration of the chiral-specific interaction in biological systems,providing potential applications for drug delivery,biosensing,and tumor detection.

Ultra-stable and multistimuli-responsive nanoparticles coated with zwitterionic pillar[n]arene for enhanced cellular uptake

Nanoparticle surface property is crucial for circulation stability,cellullar uptake and other biological characteristics.Zwitte rionic pillar[n]arenes(ZPns)we re used to coat gold nanopa rticles(GNPs)via hostguest interaction.The resulting GNPs demonstrated higher stability in blood serum compared to polyethylene glycol(PEG)-coated GNPs.ZPn-coated GNPs were responsive to UV-irradiation,competitive displacement and acidic pH.UV-irradiation or competitive displacement could lead to the removal of ZPn coating to expose GNPs,which enhanced cell uptake efficiency by 5.9-and 7.4-fold,respectively.

Effect of chirality on conformation and cellular uptake of poly(S-(o-nitrobenzyl)-L,D-cysteine) polypeptides

A series of poly（S-（o-nitrobenzyl）-L,D-cysteine） polypeptides with different chirality was synthesized and their molecular structures,secondary conformations,drug release and biological properties were thoroughly investigated.The chirality of the polypeptides had effect on secondary conformations and the cellular uptake behavior of the related nanoparticles.

Encapsulation of docosahexaenoic acid (DHA) using self-assembling food-derived proteins for efficient biological functions

Docosahexaenoic acid(DHA;22n-6)possesses multiple biological functions, including antioxidant activity and ameliorating hypertriglyceridemia. However, the application of DHA has been limited due to poor aqueous solubility and susceptible to oxidation. Here, ovalbumin(O), myosin(M), 7S soy globulin(S), and β-lactoglobulin(β), hydrolyzed by chymotrypsin, self-assembled into micelles, respectively. Adding incremental DHA to micelles caused endogenous fluorescence quenching of O, M, S, and β micelles, implying successful incorporation of DHA into hydrophobic cores of micelles(O(DHA), M(DHA), S(DHA), and β(DHA)). The results showed that micelles provided spatial stability and improved solubility, and stability against thermal and ultraviolet(UV)light for DHA. The uptake of DHA from M(DHA), β(DHA), O(DHA), and S(DHA)was 3.27-, 3.91-, 2.7-, and 3.95-fold higher, respectively, than that of DHA by Caco-2 cells. Encapsulation in micelles increased DHA aqueous solubility and uptake, which enhanced cellular endogenous antioxidant defense. Meanwhile, increased uptake of DHA was verified by HepG2 cells, and O, M, S, and β micelles were proven to increase DHA uptake to reduce lipid deposition. Our findings strongly support the possibility that O, M, S, and β micelles can be regarded as a carrier for loading DHA.

Mitochondrial transplantation strategies as potential therapeutics for central nervous system trauma

Mitochondria are essential cellular organelles critical for generating adenosine triphosphate for cellular homeostasis, as well as various mechanisms that can lead to both necrosis and apoptosis. The field of ＂mi- tochondrial medicine＂ is emerging in which injury/disease states are targeted therapeutically at the level of the mitochondrion, including specific antioxidants, bioenergetic substrate additions, and membrane uncoupling agents. Consequently, novel mitochondrial transplantation strategies represent a potentially multifactorial therapy leading to increased adenosine triphosphate production, decreased oxidative stress, mitochondrial DNA replacement, improved bioenergetics and tissue sparing. Herein, we describe briefly the history of mitochondrial transplantation and the various techniques used for both in vitro and in vivo delivery, the benefits associated with successful transference into both peripheral and central nervous system tissues, along with caveats and pitfalls that hinder the advancements of this novel therapeutic.

The adsorption of cellular proteins affects the uptake and cellular distribution of gold nanoparticles

Nanotechnology has been widely used in the field of medicine, and it can significantly improve the bioavailability and the target efficiency of medicines. However, after administration, nanomedicines can adsorb biomolecules that can influence their effects. It was reported that the adsorption of plasma proteins can change the surface properties of nanoparticles. When nanoparticles pass through cells, they may carry some cellular proteins out of cells. Currently, it is unclear whether the adsorbed proteins affect the uptake of nanoparticles in the next cell layer. To simplify this complex biological process, BSA-capped gold nanoparticles were prepared and incubated with Caco-2 cell lysate to simulate conditions of transcytosis through epithelial cells. The surface morphology of nanoparticles was examined by TEM. SRB was used to evaluate the cytotoxicity of the nanoparticles. The uptake and cellular distribution of the nanoparticles were detected by ICP-MS and CLSM. The results suggested that the adsorption of cell proteins could enhance the adhesion and uptake of gold nanoparticles. The gold nanoparticles were mainly located in lysosomes, and there were some Lysate-capped AuNPs in the mitochondria whereas no BSA-capped AuNPs appeared there.

Impact of chitosan-based nanocarriers on cytoskeleton dynamics:Current status and challenges

Chitosan-based nanocarriers(CS-NCs)show a promising role in improving drugs and bioactive compounds delivery for therapy.However,the effects exerted by CS-NCs at the cellular level,including their recognition and uptake,have not been fully investigated yet.Many factors,including size,shape,concentration,and surface chemistry of CS-NCs,play an important role in determining the types of intracellular signals triggered.The mechanism of uptake and the involvement of the cytoskeleton during the CS-NCs endocytosis variates among the different cell types as well as further effects observed inside cells.In the present work,we discuss the effects induced by CS-NCs per se on the cytoskeleton,a key component in cell architecture and physiology.The focus of this report is made on tumoral and normal biological models in which CS-NCs could differentially affect the cell cytoskeleton.The recent years reports regarding the impact of CS-NCs on cytoskeleton dynamics and the current techniques for its evaluation are summarized and discussed.Understanding mechanisms underlying cytoskeletal impact after cell exposure to CS-NCs is critical for the design of safest value-added formulations in the biomedical field.Furthermore,this revision points out some interesting aspects of cytoskeletal changes and cell death encompassing anti-tumoral effects.

Enhanced effect of dimension of receptor-ligand complex and depletion effect on receptor-mediated endocytosis of nanoparticles

We present an extended analytical model including the depletion effect and the dimension of ligand-receptor complex, aiming to elucidate their influences on endocytosis of spherocylindrical nanoparticles （NPs）. It is found that the dimension of ligand-receptor complex （δ） and the depletion effect interrelatedly govern the optimal conditions of NP endocytosis. The endocytosis phase diagram constructed in the space of NP radius and relative aspect ratio indicates that the endocytosis of NP is enhanced evidently by reducing the optimal radius and the threshold radius of endocytosed NP. Meanwhile, through thermodynamic and kinetic analysis of the diffusion of receptors, the dependence of diffusion length on depletion effect and the dimension of ligand-receptor complex can be identified in great detail. For small aspect ratio, diffusion length decreases with increasing concentration c of small bioparticles in cellular environment. Endocytosis speed corresponding to large radius R and high concentration c of small bioparticles strongly depends on the increasing （2r-δ）. These results may show some highlights into the conscious design of NPs for diagnostic agents and therapeutic drug delivery applications.

PEG400-mediated nanocarriers improve the delivery and therapeutic efficiency of mRNA tumor vaccines

Dendritic cell(DC)-targeted delivery of mRNA is a prominent method to boost the efficacy of mRNA tumor vaccines.The targeting ligands are often modified on nanocarriers by polyethylene glycol(PEG)linker in mRNA delivery systems.Whether the PEG linker length influences the targeting delivery efficiency of mRNA nanocarrier in vivo remains unclear.Here,we designed and constructed DC-targeted mRNA delivery systems modified by mannose via different PEG linker lengths(100/400/1000/2000)(MPn-LPX).The top candidate MP_(400)-LPX(the linker was PEG400)showed the optimal mRNA expression and antigen presentation owing to the highly efficient uptake by DCs.Furthermore,MP_(400)-LPX could better inhibited tumor growth and extended survival in the E.G7-OVA lymphoma and TC-1 cervical tumor mouse model.Collectively,these results demonstrated that PEG400 was the optimal linker for the PEGylated DC-targeted mRNA vaccines.Our findings provided a new platform for the rational design of targeted mRNA nanovaccines with shorter-length PEG.

Nano-bio interactions between 2D nanomaterials and mononuclear phagocyte system cells

Two-dimensional(2D)nanomaterials,known for their unique atomic arrangements and exceptional physicochemical properties,have garnered significant attention in biomedical applications,particularly in the realms of immunotherapy for tissue engineering and tumor therapy.These applications necessitate a thorough assessment of the potential influence of 2D nanomaterials on immune cells.Notably,the mononuclear phagocyte system(MPS)cells,which play pivotal roles in both innate and adaptive immunity,are essential for maintaining organismal homeostasis.MPS cells with phagocytic capability contribute to the prevention of foreign body invasion and the elimination of dead or senescent cells.Furthermore,MPS cells,including macrophages and dendritic cells,serve as vital bridges between innate and adaptive immune responses.Therefore,understanding the nano-bio interactions between 2D nanomaterials and MPS cells is imperative.These nano-bio interactions including cellular uptake,cytocompatibility,and immunological impact are invaluable forthe purposeful design of 2D nanomaterials.Herein,we provide an overview of the latest advancements in understanding the nano-bio interactions between 2D nanomaterials and MPS cells,and discuss the current challenges and future prospects of employing 2D nanomaterials in the field of nanomedicine.

Cellular arsenic transport pathways in mammals

Natural contamination of drinking water with arsenic results in the exposure of millions of people world-wide to unacceptable levels of this metalloid. This is a serious global health problem because arsenic is a Group 1（proven） human carcinogen and chronic exposure is known to cause skin, lung, and bladder tumors. Furthermore, arsenic exposure can result in a myriad of other adverse health effects including diseases of the cardiovascular,respiratory, neurological, reproductive, and endocrine systems. In addition to chronic environmental exposure to arsenic, arsenic trioxide is approved for the clinical treatment of acute promyelocytic leukemia, and is in clinical trials for other hematological malignancies as well as solid tumors. Considerable inter-individual variability in susceptibility to arsenic-induced disease and toxicity exists, and the reasons for such differences are incompletely understood. Transport pathways that influence the cellular uptake and export of arsenic contribute to regulating its cellular, tissue, and ultimately body levels. In the current review, membrane proteins（including phosphate transporters, aquaglyceroporin channels, solute carrier proteins, and ATP-binding cassette transporters） shown experimentally to contribute to the passage of inorganic, methylated, and/or glutathionylated arsenic species across cellular membranes are discussed. Furthermore, what is known about arsenic transporters in organs involved in absorption, distribution, and metabolism and how transport pathways contribute to arsenic elimination are described.

Zwitterionic peptides:Tunable next-generation stealth nanoparticle modifications

Adsorption of proteins to nanoparticles(NPs),a complex process that results in a protein corona,is controlled by NP surface properties that define NP interactions in vivo.Efforts to control adsorbed protein quantity through surface modification have led to improvements in circulation time or biodistribution.Still,current approaches have yet to be identified to control adsorbed protein identities within the corona.Here,we report the development and characterization of diverse zwitterionic peptides(ZIPs)for NP anti-fouling surface functionalization with specific and controllable affinity for protein adsorption profiles defined by ZIP sequence.Through serum exposure of ZIP-conjugated NPs and proteomics analysis of the resulting corona,we determined that protein adsorption profiles depend not on the exact composition of the ZIPs but on the sequence and order of charges along the sequence(charge motif).These findings pave the way for developing tunable ZIPs to orchestrate specific ZIP-NP protein adsorption profiles as a function of ZIP charge motif to better control cell and tissue specificity and pharmacokinetics and provide new tools for investigating relationships between protein corona and biological function.Furthermore,overall ZIP diversity enabled by the diversity of amino acids may ameliorate adaptive immune responses.

The anti-tumor efficacy of c9,t11-CLA-PTX and t10,c12-CLA-PTX on MCF-7 breast cancer cells:in vitro and in vivo

Considering the results of our previous research that conjugated linoleic acid mixture-paclitaxel （CLA-mixture-PTX） possesses anti-tumor activity against melanoma and brain glioma, the purpose of this study was to investigate the potential anti-tumor efficacy of cis-9, trans- 1 1-conjugated linoleic acid-paclitaxel （c9, tl 1-CLA-PTX） and trans- 1 O, cis- 12-conjugated linoleic acid-paclitaxel （tl0, c12-CLA-PTX） on MCF-7 breast cancer cell line in vitro and in vivo. The in vitro cytotoxicity, apoptosis induction effect and cell cycle arresting effect of c9, t1 1-CLA-PTX and t10, c12-CLA-PTX were investigated. The in vitro cellular uptake of c9, tl 1-CLA-PTX and tl0, cl2-CLA-PTX in MCF-7 cells were also analyzed. Besides, the anti-tumor activity of c9, tl 1-CLA-PTX and tl0, cl2-CLA-PTX was evaluated in MCF-7 tumor bearing nude mice in vivo. The in vitro cytotoxicity results showed that the value of ICs0 of the tl 0, c l2-CLA-PTX is （0.17±0.02） μM, compared with that of （1.08±0.15） μM in CLA-mixture-PTX and （6.50±1.20） μM in c9, tl 1-CLA-PTX treatment group （P〈0.01）. Both tl0, cl2-CLA-PTX and c9, t l 1-CLA-PTX increased the percentage of total apoptotic cells compared with that of control （P〈0.01）. And the rank of apoptosis induction efficacy was t 10, c 12-CLA-PTX〉CLA-mixture-PTX〉c9, t 11-CLA-PTX （P〈0.01）. Compared with untreated cells, the tl0, c12-CLA-PTX and c9, tl 1-CLA-PTX arrested cell cycle progression at the S and G2-M phase. The amount of cellular uptake of t 10, c 12-CLA-PTX was significantly higher than that of CLA-mixture-PTX （P〈0.01）, which was significantly higher than that of c9, t1 1-CLA-PTX （P〈0.01）. The rank of in vivo anti-tumor activity was tl0, c12-CLA-PTX〉CLA-mixture-PTX〉 c9, t1 1-CLA-PTX （P〈0.01）. In conclusion, our study demonstrated that both tl0, cl2-CLA-PTX and c9, tl 1-CLA-PTX has significant anti-tumor activity in MCF-7 cell line. And while c9, tl 1-CLA-PTX showed weaker inhibitory effect than CLA-mixture-PTX, stronger inhibitory effect was presented by t10, c12-CLA-PTX, which could be a promising alternative for CLA-mixture-PTX.

In vitro and in vivo antitumor efficacy of CLA-PTX on B16-F10 melanoma cells

The purpose of this study was to investigate the potential antitumor efficacy of conjugated linoleic acid-paclitaxel （CLA-PTX） on B16-F10 melanoma cell line in vitro and in vivo. The in vitro cytotoxicity, apoptosis and cell cycle of CLA-PTX were investigated. The in vitro cellular uptake of CLA-PTX in B16-F10 cells was also analyzed. The antitumor activity of CLA-PTX was also evaluated in B16-F10 tumor-bearing C57BL6/N mice in vivo. The in vitro cytotoxicity results showed that the IC50 of the CLA-PTX is （4.25±0.43） μM, compared with that of （6.70±0.80） μM in PTX treatment group （P〈0.01）. CLA-PTX increased the percentage of total apoptotic cells compared with that of control and PTX treatment groups （P〈0.01）. Compared with untreated cells, CLA-PTX arrested cell cycle progression at the S phase, whereas PTX caused accumulation of cell at GE-M phase both along with the reduction of the cellular fraction arrested at the G1 phase. The amount of cellular uptake of CLA-PTX was significantly higher than that of PTX （P〈0.01）. The in vivo antitumor activity of CLA-PTX was significantly higher than that of control and PTX treatment groups （P〈0.01 or P〈0.05）. In conclusion, our study demonstrated that CLA-PTX has significant antitumor activity in B 16-F 10 cell line.

Pulmonary delivery of siRNA against acute lung injury/acute respiratory distress syndrome

The use of small interfering RNAs(si RNAs)has been under investigation for the treatment of several unmet medical needs,including acute lung injury/acute respiratory distress syndrome(ALI/ARDS)wherein si RNA may be implemented to modify the expression of pro-inflammatory cytokines and chemokines at the m RNA level.The properties such as clear anatomy,accessibility,and relatively low enzyme activity make the lung a good target for local si RNA therapy.However,the translation of si RNA is restricted by the inefficient delivery of si RNA therapeutics to the target cells due to the properties of naked si RNA.Thus,this review will focus on the various delivery systems that can be used and the different barriers that need to be surmounted for the development of stable inhalable si RNA formulations for human use before si RNA therapeutics for ALI/ARDS become available in the clinic.