Myosin 9 and N-glycans jointly regulate human papillomavirus entry.

Persistent high-risk HPV infection is closely associated with cervical cancer development, and there is no drug targeting HPV on the market at present, so it is particularly important to understand the interaction mechanism between HPV and the host which may provide the novel strategies for treating HPV diseases. HPV can hijack cell surface heparan sulfate proteoglycans (HSPGs) as primary receptors. However, the secondary entry receptors for HPV remain elusive. We identify myosin-9 (NMHC-IIA) as a host factor that interacts with HPV L1 protein and mediates HPV internalization. Efficient HPV entry required myosin-9 redistribution to the cell surface regulated by HPV-hijacked MEK-MLCK signaling. Myosin-9 maldistribution by ML-7 or ML-9 significantly inhibited HPV pseudoviruses infection in vitro and in vivo. Meanwhile, N-glycans, especially the galactose chains, may act as the decoy receptors for HPV, which can block the interaction of HPV to myosin-9 and influence the way of HPV infection. Taken together, we identify myosin-9 as a novel functional entry receptor for high-risk HPV both in vitro and in vivo, and unravel the new roles of myosin-9 and N-glycans in HPV entry, which provides the possibilities for host targets of antiviral drugs.

Progesterone-Cationic Lipid Conjugate-Based Self-Aggregates for Cancer Cell-Selective Uptake through Macropinocytosis and the Antitumour Effect.

Progesterone (PR) is an endogenous steroid hormone that activates the progesterone receptor (PgR) and is known to play a critical role in cancer progression. Herein, we report the development of cationic lipid-conjugated PR derivatives by covalently conjugating progesterone with cationic lipids of varying hydrocarbon chain lengths (n = 6-18) through a succinate linker. Cytotoxicity studies performed on eight different cancer cell lines reveal that PR10, one of the lead derivatives, exerts notable toxicity (IC50 = 4-12 µM) in cancer cells irrespective of their PgR expression status and remains largely nontoxic to noncancerous cells. Mechanistic studies show that PR10 induces G2/M-phase cell cycle arrest in cancer cells, leading to apoptosis and cell death by inhibiting the PI3K/AKT cell survival pathway and p53 upregulation. Further, in vivo study shows that PR10 treatment significantly reduces melanoma tumor growth and prolongs the overall survival of melanoma tumor-bearing C57BL/6J mice. Interestingly, PR10 readily forms stable self-aggregates of ∼190 nm size in an aqueous environment and exhibits selective uptake into cancerous cell lines. In vitro uptake mechanism studies in various cell lines (cancerous cell lines B16F10, MCF7, PC3, and noncancerous cell line HEK293) using endocytosis inhibition proves that PR10 nanoaggregates enter selectively into the cancer cells predominantly using macropinocytosis and/or caveolae-mediated endocytosis. Overall, this study highlights the development of a self-aggregating cationic derivative of progesterone with anticancer activity, and its cancer cell-selective accumulation in nanoaggregate form holds great potential in the field of targeted drug delivery.

Internalization and membrane activity of the antimicrobial peptide CGA-N12.

Antimicrobial peptides (AMPs) are conventional antibiotic alternatives due to their broad-spectrum antimicrobial activities and special mechanisms of action against pathogens. The antifungal peptide CGA-N12 was originally derived from human chromogranin A (CGA) and consists of the 65th to 76th amino acids of the CGA N-terminal region. In the present study, we found that CGA-N12 had fungicidal activity and exhibited time-dependent inhibition activity against Candida tropicalis. CGA-N12 entered the cells to exert its antagonist activity. The internalization of CGA-N12 was energy-dependent and accompanied by actin cytoskeleton-, clathrin-, sulfate proteoglycan-, endosome-, and lipid-depleting agent-mediated endocytosis. Moreover, the CGA-N12 internalization pathway was related to the peptide concentration. The effects of CGA-N12 on the cell membrane were investigated. CGA-N12 at a low concentration less than 4 × MIC100 did not destroy the cell membrane. While with increasing concentration, the damage to the cell membrane caused by CGA-N12 became more serious. At concentrations greater than 4 × MIC100, CGA-N12 destroyed the cell membrane integrity. Therefore, the membrane activity of CGA-N12 is concentration dependant.

Microfluidic-Based Cationic Cholesterol Lipid siRNA Delivery Nanosystem: Highly Efficient In Vitro Gene Silencing and the Intracellular Behavior.

Safe and efficient delivery of small interfering RNA (siRNA) is essential to gene therapy towards intervention of genetic diseases. Herein, we developed a novel cationic cholesterol lipid derivative (CEL) in which cholesterol hydrophobic skeleton was connected to L-lysine cationic headgroup via a hexanediol linker as the non-viral siRNA delivery carrier. Well-organized CEL/siRNA nanocomplexes (100-200 nm) were prepared by microfluidic-assisted assembly of CEL and siRNA at various N/P ratios. The CEL and CEL/siRNA nanocomplexes have lower cytotoxicity compared with bPEI25k. Delightfully, we disclosed that, in Hela-Luc and H1299-Luc cell lines, the micro-fluidic-based CEL/siRNA nanocomplexes exhibited high siRNA transfection efficiency under both serum-free condition (74-98%) and low-serum circumstances (80-87%), higher than that of lipofectamine 2000. These nanocomplexes also showed high cellular uptake through the caveolae/lipid-raft mediated endocytosis pathway, which may greatly contribute to transfection efficiency. Moreover, the time-dependent (0-12 h) dynamic intracellular imaging demonstrated the efficient delivery to cytoplasm after lysosomal co-localization. The results indicated that the microfluidic-based CEL/siRNA nanosystems possessed good stability, low cytotoxicity, high siRNA delivery efficiency, rapid cellular uptake and caveolae/lipid raft-dependent internalization. Additionally, this study provides a simple approach for preparing and applying a "helper lipid-free" cationic lipid siRNA delivery system as potential nanotherapeutics towards gene silencing treatment of (tumor) diseases.

Evaluation of size, shape, and charge effect on the biological interaction and cellular uptake of cerium oxide nanostructures.

Cerium oxide (CeO2) at the nanoscale has prolifically attracted the immense interest of researchers due to its switchable oxidation states (Ce3+/Ce4+) that play a crucial role in many biological activities. The present work reports the evaluation of size, shape, and charge effect on the biological interaction with RAW 264.7 cells for three nanostructures of CeO2(CeO2NS) namely nanocubes (NCs), nanorods (NRs), and nanoparticles (NPs). These NS exhibits similar composition and have average diameter values in the order of NCs < NRs â‰… NPs. The values of zeta potential revealed the anionic nature of NS with surface charge in order of NCs < NPs < NRs. The cellular interaction of CeO2NS was analyzed for cytotoxicity, cellular uptake, and morphological studies. Quantitative determination of the uptake of CeO2NS exhibited concentration-dependent uptake in the order as NCs > NPs > NRs. The proposed possible mechanisms of cellular uptake revealed that different structures tended to use the various endocytosis pathways in different proportions.

Skeleton-Controlled pDNA Delivery of Renewable Steroid-Based Cationic Lipids, the Endocytosis Pathway Analysis and Intracellular Localization.

Using renewable and biocompatible natural-based resources to construct functional biomaterials has attracted great attention in recent years. In this work, we successfully prepared a series of steroid-based cationic lipids by integrating various steroid skeletons/hydrophobes with (l-)-arginine headgroups via facile and efficient synthetic approach. The plasmid DNA (pDNA) binding affinity of the steroid-based cationic lipids, average particle sizes, surface potentials, morphologies and stability of the steroid-based cationic lipids/pDNA lipoplexes were disclosed to depend largely on the steroid skeletons. Cellular evaluation results revealed that cytotoxicity and gene transfection efficiency of the steroid-based cationic lipids in H1299 and HeLa cells strongly relied on the steroid hydrophobes. Interestingly, the steroid lipids/pDNA lipoplexes inclined to enter H1299 cells mainly through caveolae and lipid-raft mediated endocytosis pathways, and an intracellular trafficking route of "lipid-raft-mediated endocytosis→lysosome→cell nucleic localization" was accordingly proposed. The study provided possible approach for developing high-performance steroid-based lipid gene carriers, in which the cytotoxicity, gene transfection capability, endocytosis pathways, and intracellular trafficking/localization manners could be tuned/controlled by introducing proper steroid skeletons/hydrophobes. Noteworthy, among the lipids, Cho-Arg showed remarkably high gene transfection efficacy, even under high serum concentration (50% fetal bovine serum), making it an efficient gene transfection agent for practical application.

Valproic Acid Induces Endocytosis-Mediated Doxorubicin Internalization and Shows Synergistic Cytotoxic Effects in Hepatocellular Carcinoma Cells.

Valproic acid (VPA), a well-known histone deacetylase (HDAC) inhibitor, is used as an anti-cancer drug for various cancers, but the synergistic anti-cancer effect of VPA and doxorubicin (DOX) combination treatment and its potential underlying mechanism in hepatocellular carcinoma (HCC) remain to be elucidated. Here, we evaluate the mono- and combination-therapy effects of VPA and DOX in HCC and identify a specific and efficient, synergistic anti-proliferative effect of the VPA and DOX combination in HCC cells, especially HepG2 cells; this effect was not apparent in MIHA cells, a normal hepatocyte cell line. The calculation of the coefficient of drug interaction confirmed the significant synergistic effect of the combination treatment. Concurrently, the synergistic apoptotic cell death caused by the VPA and DOX combination treatment was confirmed by Hoechst nuclear staining and Western blot analysis of caspase-3 and poly (ADP-ribose) polymerase (PARP) activation. Co-treatment with VPA and DOX enhanced reactive oxygen species (ROS) generation and autophagy, which were clearly attenuated by ROS and autophagy inhibitors, respectively. Furthermore, as an indication of the mechanism underlying the synergistic effect, we observed that DOX internalization, which was induced in the VPA and DOX combination-treated group, occurred via by the caveolae-mediated endocytosis pathway. Taken together, our study uncovered the potential effect of the VPA and DOX combination treatment with regard to cell death, including induction of cellular ROS, autophagy, and the caveolae-mediated endocytosis pathway. Therefore, these results present novel implications in drug delivery research for the treatment of HCC.

Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin.

Heat-labile enterotoxin (LT) is a protein toxin produced by enterotoxigenic Escherichia coli (ETEC). As a bacterial toxin, LT holotoxin can enter intestinal epithelial cells and cause diarrhea. In addition, LT is also a powerful mucosal adjuvant capable of enhancing the strong immune responses to co-administered antigens. However, the LT immunological mechanism is still not clear in some aspects, especially with the respect to how the LTA subunit functions alone. Here, we discovered that the A2 domain of LTA could carry a fluorescent protein into cells, whose function is similar to a cell-penetrating peptide. The transmembrane-transporting ability of the A2 domain is non-specific in its cell-penetrating function, which was shown through testing with different cell types. Moreover, the LTA2 fusion protein penetrated a fluorescently labeled cell membrane that identified LTA2 internalization through membrane transport pathways, and showed it finally localized in the endoplasmic reticulum. Furthermore, low-temperature stress and pharmacological agent treatments showed that the LTA2 internalization route is a temperature-dependent process involving the clathrin-mediated endocytosis and the macropinocytosis pathways. These results could explain the internalization of the LTA subunit alone without the LTB pentamer, contributing to a better understanding of LTA working as a mucosal adjuvant; they also suggest that the A2 domain could be used as a novel transport vehicle for research and treatment of disease.

Cellular uptake mechanism of TCTP-PTD in human lung carcinoma cells.

We reported previously that human translationally controlled tumor protein (TCTP) contains, at its NH2-terminus, a protein transduction domain (PTD), which we called TCTP-PTD, with the amino acid sequence MIIYRDLISH. In this report we describe how TCTP-PTD penetrates A549 human lung cancer cell membranes and promotes protein internalization. Cellular uptake of fluorescent TCTP-PTD and a recombinant fusion protein consisting of TCTP-PTD and GFP (green fluorescent protein) was analyzed by confocal fluorescence microscopy and flow cytometry. Inhibitor assays using several agents that perturb the internalization process revealed that TCTP-PTD transduces the cells partly via lipid-raft/caveola-dependent endocytosis and partly by macropinocytosis in a dynamin/actin/microtubule-dependent pathway. To trace the pathway followed by the penetration of TCTP-PTD, the localization of PTDs was investigated in the lipid-raft, subcellular, and ER fractions. We found that, after entry, TCTP-PTD is localized in the cytoplasm and cytoskeleton, but not in the nucleus, and is transported into endoplasmic reticulum (ER). Expression levels of caveolin-1 in A549 and HeLa cells are different, and these differences appear to contribute to the sensitivity of TCTP-PTD uptake inhibition, against lipid-raft depleter, nystatin. This elucidation of the underlying mechanism of TCTP-PTD translocation may help the design of approaches that employ TCTP-PTD in the cellular delivery of bioactive molecules.

Global adaptive rank truncated product method for gene-set analysis in association studies.

Gene set analysis (GSA) aims to assess the overall association of a set of genetic variants with a phenotype and has the potential to detect subtle effects of variants in a gene or a pathway that might be missed when assessed individually. We present a new implementation of the Adaptive Rank Truncated Product method (ARTP) for analyzing the association of a set of Single Nucleotide Polymorphisms (SNPs) in a gene or pathway. The new implementation, referred to as globalARTP, improves the original one by allowing the different SNPs in the set to have different modes of inheritance. We perform a simulation study for exploring the power of the proposed methodology in a set of scenarios with different numbers of causal SNPs with different effect sizes. Moreover, we show the advantage of using the gene set approach in the context of an Alzheimer's disease case-control study where we explore the endocytosis pathway. The new method is implemented in the R function globalARTP of the globalGSA package available at http://cran.r-project.org.

Investigation of cellular uptake and transport capacity of Cordyceps sinensis exopolysaccharide­selenium nanoparticles with different particle sizes in Caco-2 cell monolayer.

Cordyceps sinensis exopolysaccharide­selenium nanoparticles (EPS-SeNPs) were successfully constructed, characterized, and its Se release kinetics and mechanism were also evaluated in our previous studies. However, the intestinal cellular uptake and transport capacities of EPS-SeNPs remain unknown. On the basis of our previous researches, this work was designed to evaluate the uptake and transport capacities of EPS-SeNPs (EPS/Se = 20/1, 3/1, 1/1, and 3/4) in intestinal epithelial (Caco-2) cells. Confocal laser scanning microscopy results indicated that the internalization of coumarin-6 labeled EPS-SeNPs was in a time-dependent process and eventually located in the cytoplasm, not in the nucleus. Endocytosis inhibitors were employed to evaluate the cellular uptake pathway of EPS-SeNPs, relevant results revealed that clathrin-, caveolae-, and energy-mediated pathways were participated in the internalization of EPS-SeNPs by Caco-2 cells. In addition, the transportation of EPS-SeNPs across Caco-2 cell monolayers was in a concentration-dependent manner. Different particle sizes of EPS-SeNPs presented different uptake and transport capacities in Caco-2 cells. Noteworthy, EPS/Se = 3/4 with the highest selenium content possessed the most superior cellular uptake and transport abilities in Caco-2 cells. The present work may contribute to illustrate the internalization and transport mechanism of EPS-SeNPs, thus facilitating its application in food and medical industries.

Diffusion coefficient of cationic liposomes during lipoplex formation determines transfection efficiency in HepG2 cells.

Cationic lipid-based lipoplexes are well-known for gene delivery. To determine the relationship between physicochemical characteristics and transfection efficiency, cationic liposomes of different sizes were prepared and incubated with plasmid DNA at different temperatures to form lipoplexes. We found that the liposome diffusion coefficient during lipoplex formation strongly correlated with the physicochemical characteristics of lipoplexes, accessibility of plasmid DNA in lipoplexes, and logarithm of gene expression per metabolic activity. Clathrin-mediated endocytosis was the major route for lipoplexes comprising 100 nm-liposomes, as reported previously. As liposome size increased, the major route shifted to lipid raft-mediated endocytosis. In addition, macropinocytosis was observed for all liposome sizes. The role of reactive oxygen species might depend on liposome size and endocytosis. Information from this study would be useful for understanding cationic lipoplex-mediated transfection.

Intracellular fate and immune response of porphyrin-based nano-sized metal-organic frameworks.

The diverse applications of porphyrin-based nano-sized metal-organic frameworks (NMOFs) lead to great exposure risks to human and environment. Understanding the cellular biological effects (such as toxicity, distribution, and localization) of porphyrinic NMOFs is a prerequisite to the assessment of their health risk. However, the characteristics of distribution, localization, and immune response induced by porphyrinic NMOFs have not been studied yet. Here, we report the size-dependent biological effects of porphyrinic NMOFs under sublethal dose. Various sizes of PCN-224 (30, 90, and 180 nm) were taken as model porphyrinic NMOFs. We found that 30 nm PCN-224 gave the highest uptake content, followed by 90 and 180 nm PCN-224. The mechanism for uptake was clathrin-mediated for 30 and 90 nm PCN-224, but clathrin- and glycosylphosphatidylinositol-mediated for 180 nm PCN-224. All PCN-224 were localized in lysosome with size-dependent velocity of colocalization transport. 30 nm PCN-224 induced the highest released cytokines than 90 and 180 nm PCN-224 accompanied with the activation of NF-κB pathway. This work reveals the mechanisms for the endocytosis of PCN-224 and the release of cytokine induced by PCN-224, which is helpful for the health risk assessment of NMOFs.

The Rabep1-Mediated Endocytosis and Activation of Trypsinogen to Promote Pancreatic Stellate Cell Activation.

BACKGROUND: The pathogenesis of chronic pancreatitis is still unclear. Trypsinogen activation is an active factor in acute pancreatitis that has not been studied in the occurrence of chronic pancreatitis. METHODS: Immunofluorescence was used to detect the location and expression of trypsinogen in chronic pancreatitis and normal tissues. Microarray and single-cell RNA-seq (scRNA-seq) were used to screen core genes and pathways in pancreatic stellate cells (PSCs). Western blotting and immunofluorescence were used to verify trypsinogen expression in PSCs after silencing Rabep1. Immunofluorescence and flow cytometry were used to validate trypsinogen activation and PSC activation after intervening in the endocytosis pathway. RESULTS: Endocytosed trypsinogen was found in PSCs in CP clinical samples. Bioinformatic analysis showed that Rabep1 is a core gene that regulates trypsinogen endocytosis through the endocytosis pathway, verified by Western blot and immunofluorescence. Immunofluorescence and flow cytometry analyses confirmed the activation of trypsinogen and PSCs through the endocytosis pathway in PSCs. CONCLUSION: This study discovered a new mechanism by which trypsinogen affects the activation of PSCs and the occurrence and development of CP. Through communication between pancreatic acinar cells and PSCs, trypsinogen can be endocytosed by PSCs and activated by the Rabep1 gene.

Endocytosis and Organelle Targeting of Nanomedicines in Cancer Therapy.

Nanomedicines (NMs) have played an increasing role in cancer therapy as carriers to efficiently deliver therapeutics into tumor cells. For this application, the uptake of NMs by tumor cells is usually a prerequisite to deliver the cargo to intracellular locations, which mainly relies on endocytosis. NMs can enter cells through a variety of endocytosis pathways. Different endocytosis pathways exhibit different intracellular trafficking routes and diverse subcellular localizations. Therefore, a comprehensive understanding of endocytosis mechanisms is necessary for increasing cellular entry efficiency and to trace the fate of NMs after internalization. This review focuses on endocytosis pathways of NMs in tumor cells, mainly including clathrin- and caveolae-mediated endocytosis pathways, involving effector molecules, expression difference of those molecules between normal and tumor cells, as well as the intracellular trafficking route of corresponding endocytosis vesicles. Then, the latest strategies for NMs to actively employ endocytosis are described, including improving tumor cellular uptake of NMs by receptor-mediated endocytosis, transporter-mediated endocytosis and enabling drug activity by changing intracellular routes. Finally, active targeting strategies towards intracellular organelles are also mentioned. This review will be helpful not only in explicating endocytosis and the trafficking process of NMs and elucidating anti-tumor mechanisms inside the cell but also in rendering new ideas for the design of highly efficacious and cancer-targeted NMs.

Post-production modifications of murine mesenchymal stem cell (mMSC) derived extracellular vesicles (EVs) and impact on their cellular interaction.

In regards to their key role in intercellular communication, extracellular vesicles (EVs) have a strong potential as bio-inspired drug delivery systems (DDS). With the aim of circumventing some of their well-known issues (production yield, drug loading yield, pharmacokinetics), we specifically focused on switching the biological vision of these entities to a more physico-chemical one, and to consider and fine-tune EVs as synthetic vectors. To allow a rational use, we first performed a full physico-chemical (size, concentration, surface charge, cryoTEM), biochemical (western blot, proteomics, lipidomics, transcriptomics) and biological (cell internalisation) characterisation of murine mesenchymal stem cell (mMSC)-derived EVs. A stability study based on evaluating the colloidal behaviour of obtained vesicles was performed in order to identify optimal storage conditions. We evidenced the interest of using EVs instead of liposomes, in regards to target cell internalisation efficiency. EVs were shown to be internalised through a caveolae and cholesterol-dependent pathway, following a different endocytic route than liposomes. Then, we characterised the effect of physical methods scarcely investigated with EVs (extrusion through 50 nm membranes, freeze-drying, sonication) on EV size, concentration, structure and cell internalisation properties. Our extensive characterisation of the effect of these physical processes highlights their promise as loading methods to make EVs efficient delivery vehicles.

Identification of Novel Macropinocytosing Human Antibodies by Phage Display and High-Content Analysis.

Internalizing antibodies have great potential for the development of targeted therapeutics. Antibodies that internalize via the macropinocytosis pathway are particularly promising since macropinocytosis is capable of mediating rapid, bulk uptake and is selectively upregulated in many cancers. We hereby describe a method for identifying antibodies that internalize via macropinocytosis by screening phage-displayed single-chain antibody selection outputs with an automated fluorescent microscopy-based high-content analysis platform. Furthermore, this method can be similarly applied to other endocytic pathways if other fluorescent, pathway-specific, soluble markers are available.

The key molecular events during Macrobrachium rosenbergii nodavirus (MrNV) infection and replication in Sf9 insect cells.

In this study we demonstrated that Macrobrachium rosenbergii nodavirus (MrNV) was able to internalize and replicate in Sf9 insect cells, with levels of infection altered by substances affecting the caveolin-(CAV) mediated endocytosis pathway. The use of Sf9 cells for efficient MrNV replication and propagation was demonstrated by confocal microscopy and PCR amplification, through which early viral binding and internalization were initially detectable at 30min post-infection; whereas at 72h, the distinguishable sign of late-MrNV infection was observable as the gradual accumulation of a cytopathic effect (CPE) in the cells, ultimately resulting in cellular disruption. Moreover, during the early period of infection, the MrNV signals were highly co-localized with CAV1 signals of the CAV-mediated endocytosis pathway. The use of genistein as an inhibitor of the CAV-mediated endocytosis pathway significantly reduced MrNV and CAV1 co-localization, and also reduced the levels of MrNV infection in Sf9 cells as shown by PCR and ELISA. Moreover, the addition of the pathway agonist okadaic acid not only recovered but also augmented both the levels of MrNV co-localization with CAV1 and of Sf9 infection in the presence of genistein inhibition; therefore demonstrating that MrNV infection in Sf9 cells was associated with the CAV-mediated endocytosis pathway machinery.

Achieving high gene delivery performance with caveolae-mediated endocytosis pathway by (l)-arginine/(l)-histidine co-modified cationic gene carriers.

Developing new amphiphilic polymers with natural product moieties has been regarded as a promising way to achieve biocompatibility and certain biological functions. In prior work, we developed some natural (l)-arginine modified cationic polymers (PAHMAA-Rs) as cationic gene carriers. For the sake of continuing optimize the gene delivery performance, herein, a new series of (l)-arginine and (l)-histidine co-modified cationic poly (ω-aminohexyl methacrylamide)s (PAHMAA-R-H) were synthesized and characterized with 1H NMR, GPC-SLS and FT-IR. Their proton buffering capacities were studied by acid-base titration assay. pDNA binding affinity and self-assembly properties of the polyplexes were analyzed by agarose gel retardation assay, DLS and AFM, respectively. In vitro cytotoxicity of the PAHMAA-R-H was determined by MTT and LDH assays in H1299 cells, the gene transfection efficacy and intracellular uptake capability were evaluated by luciferase assay and FACS, respectively. Moreover, the endocytosis pathways and intracellular distribution of the polyplexes were investigated by using specific endocytic inhibitors and fluorescent co-localization techniques. The results demonstrated that co-modification of (l)-arginine and (l)-histidine onto the PAHMAA polymer could enhance proton buffering capacity, shield surface charge, decrease cytotoxicity, and improve gene transfection efficiency and serum-compatibility. Moreover, the gene transfection and intracellular uptake behaviors were disclosed strongly rely on the (l)-arginine/(l)-histidine modification ratios. The polyplexes tend to be internalized through caveolae-mediated endocytosis gateway and localized with endosomes/lysosomes in H1299 cells. Notably, among the polymers, the PAHMAA-R18-H6 exhibited remarkable gene delivery efficiency and serum compatibility, which made it promising gene transfection agent for practical application.

Intracellular plasmid DNA delivery by self-assembled nanoparticles of amphiphilic PHML-b-PLLA-b-PHML copolymers and the endocytosis pathway analysis.

This work presents a new series of polycationic nanoparticles of (l-)-lysine conjugated amphiphilic triblock copolymer poly(hydroxyletheyl methacrylate-L-lysine)-b-poly(L-lactide)-b-poly(hydroxyletheyl methacrylate-L-lysine)s (PHML-b-PLLA-b-PHML) as potent low cytotoxic vectors for intracellular plasmid DNA delivery. First, the triblock PHML-b-PLLA-b-PHML copolymers were prepared via a combination of metal-free controlled ring opening polymerization and successive atom transfer radical polymerization. Then the cationic PHML-b-PLLA-b-PHML nanoparticles were further prepared by solution self-assembly. The particle size, zeta potential and morphology of as-prepared PHML-b-PLLA-b-PHML nanoparticles were characterized by dynamic light scattering and atomic force microscopy, respectively. The plasmid DNA binding affinities and polyplex stabilities were separately explored by agarose gel retardation and DNase I degradation assays. Then in vitro cytotoxicity and gene transfection efficiency of the PHML-b-PLLA-b-PHML nanoparticles vectors as well as relevant polyplex endocytosis pathway were investigated with H1299 cells. It was revealed that the PHML-b-PLLA-b-PHML nanoparticles exhibited low cytotoxicity, strong plasmid DNA binding affinity, high polyplex stability and efficient plasmid DNA transfection even under serum conditions (10% FBS). Moreover, the endocytosis analysis results disclosed that the PHML30-b-PLLA-b-PHML30 nanoparticle/plasmid DNA polyplexes were predominantly involved in lipid-raft-mediated endocytosis pathway, similar to that of SV40 virus-based vectors.