Cell-penetrating peptides with nanoparticles hybrid delivery vectors and their uptake pathways.

Cell-penetrating peptides (CPPs) are molecules that improve the cellular uptake of various molecular payloads that do not easily traverse the cellular membrane. CPPs can be found in pharmaceutical and medical products. The vast majority of cell-penetrating chemicals that are discussed in published research are peptide based. The paper also delves into the various applications of hybrid vectors. Because CPPs are able to carry cargo across the cellular membrane, they are a viable candidate for use as a suitable carrier for a wide variety of cargoes, such as siRNA, nanoparticles, and others. In which we discuss the CPPs, their classification, uptake mechanisms, hybrid vector systems, nanoparticles and their uptake mechanisms, etc. Further in this paper, we discuss CPPs conjugated to Nanoparticles, Combining CPPs with lipids and polymeric Nanoparticles in A Conjugated System, CPPs conjugated to nanoparticles for therapeutic purposes, and potential therapeutic uses of CPPs as delivery molecules. Also discussed the preclinical and clinical use of CPPS, intracellular trafficking of nanoparticles, and activatable and bioconjugated CPPs.

[Evaluation of Solubility and Membrane Permeability of Middle-Molecule Compounds Using Artificial Membranes and Living Cells].

In contrast to small molecules, middle molecules present a promising therapeutic modality owing to their elevated specificity, minimal adverse effects, capacity to target protein-protein interactions, and, unlike antibody-based drugs, their suitability for oral administration and intracellular target engagement. Post-oral administration, the paramount considerations encompass solubility and membrane permeability during the initial phase until the drug attains systemic circulation. Furthermore, penetration of the cell membrane is essential to accessing intracellular targets. We evaluated the solubility and membrane permeability of 965 compounds sourced from middle molecule libraries affiliated with Hokkaido University, Kitasato University, and the University of Tokyo. To gauge membrane permeability, we employed both the parallel artificial membrane permeability assay (PAMPA) and Caco-2 cell monolayers. Notably, while membrane permeability in Caco-2 cells exhibited an approximate threefold increase in comparison to PAMPA measurements, certain compounds demonstrated permeability levels less than one-third of those observed in Caco-2 cells. Recognizing the potential involvement of efflux transporters expressed in Caco-2 cells in these variations, we conducted additional assessments involving directional transport in the presence of a transporter inhibitor. Our findings suggest that nearly 80% of these compounds serve as substrates for efflux transporters. Considering the relevance of intracellular targets, we shifted our focus from membrane permeation to intracellular uptake, conducting simulations tailored to assess cellular uptake.

Engineered Hybrid Vesicles and Cellular Internalization in Mammary Cancer Cells.

Extracellular vesicles play an important role in intercellular communication, with the potential to serve as biomaterials for nanocarriers. Combining such extracellular vesicles and liposomes results in advanced drug delivery carriers. In this study, we attempted to fabricate hybrid vesicles using a membrane fusion method and incorporated an anticancer drug. As a result, we successfully prepared nanosized uniform hybrid vesicles and evaluated their physicochemical characteristics and intracellular uptake mechanisms via endocytosis in various cell lines. Compared to liposomes, the hybrid vesicles showed better physical properties and a relatively higher reduction in cell viability, which was presumably dependent on the specific cell type. These findings suggest that fusion-based hybrid vesicles offer a novel strategy for delivering therapeutic agents and provide insights into the types of extracellular vesicles that are useful in fabricating hybrid vesicles to develop an advanced drug delivery system.

Magnetically Enhanced Intracellular Uptake of Superparamagnetic Iron Oxide Nanoparticles for Antitumor Therapy.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely employed in biomedical fields, including targeted delivery of antitumor therapy. Conventional magnetic tumor targeting has used simple static magnetic fields (SMFs), which cause SPIONs to linearly aggregate into a long chain-like shape. Such agglomeration greatly hinders the intracellular targeting of SPIONs into tumors, thus reducing the therapeutic efficacy. In this study, we investigated the enhancement of the intracellular uptake of SPIONs through the application of rotating magnetic fields (RMFs). Based on the physical principles of SPION chain disassembly, we investigated physical parameters to predict the chain length favorable for intracellular uptake. Our prediction was validated by clear visualization of the intracellular distributions of SPIONs in tumor cells at both cellular and three-dimensional microtissue levels. To identify the potential therapeutic effects of enhanced intracellular uptake, magnetic hyperthermia as antitumor therapy was investigated under varying conditions of magnetic hyperthermia and RMFs. The results showed that enhanced intracellular uptake reduced magnetic hyperthermia time and strength as well as particle concentration. The proposed method will be useful in the development of techniques to determine the optimized physical conditions for the enhanced intracellular uptake of SPIONs in antitumor therapy.

Stereoisomer-Dependent Membrane Association and Capacity for Insulin Delivery Facilitated by Penetratin.

Cell-penetrating peptides (CPPs), such as penetratin, are often investigated as drug delivery vectors and incorporating d-amino acids, rather than the natural l-forms, to enhance proteolytic stability could improve their delivery efficiency. The present study aimed to compare membrane association, cellular uptake, and delivery capacity for all-l and all-d enantiomers of penetratin (PEN) by using different cell models and cargos. The enantiomers displayed widely different distribution patterns in the examined cell models, and in Caco-2 cells, quenchable membrane binding was evident for d-PEN in addition to vesicular intracellular localization for both enantiomers. The uptake of insulin in Caco-2 cells was equally mediated by the two enantiomers, and while l-PEN did not increase the transepithelial permeation of any of the investigated cargo peptides, d-PEN increased the transepithelial delivery of vancomycin five-fold and approximately four-fold for insulin at an extracellular apical pH of 6.5. Overall, while d-PEN was associated with the plasma membrane to a larger extent and was superior in mediating the transepithelial delivery of hydrophilic peptide cargoes compared to l-PEN across Caco-2 epithelium, no enhanced delivery of the hydrophobic cyclosporin was observed, and intracellular insulin uptake was induced to a similar degree by the two enantiomers.

Cellular uptake of rose bengal is mediated by OATP1B1/1B3 transporters.

Due to its fluorescent properties and high yield of singlet oxygen, rose bengal (RB) is one of the most promising photosensitizers for cancer treatment. However, the negative charge of RB molecule may significantly hamper its intracellular delivery by passive diffusion through the cell membrane. Thus, specific membrane protein transporters may be needed. The organic anion transporting polypeptides (OATPs) are a well-characterized group of membrane protein transporters, responsible for cellular uptake of a number of drugs. To our knowledge, this is the first study that evaluates cellular transport of RB mediated by the OATP transporter family. First, electrified liquid-liquid interface, together with biophysical analysis and molecular dynamics simulations were used to characterize the interaction of RB with several models of a cellular membranes. These experiments proved that RB interacts only with the membrane's surface, without spontaneously crossing the lipid bilayer. Evaluation of intracellular uptake of RB by flow cytometry and confocal microscopy showed significant differences in uptake between liver and intestinal cell line models differing in expression of OATP transporters. The use of specific pharmacological inhibitors of OATPs, together with Western blotting and in silico analysis, indicated that OATPs are crucial for cellular uptake of RB.

A rapid intracellular enrichment of alkylating payload is essential for melphalan flufenamide potency and mechanism of action.

Despite decades of development of treatments and the successful application of targeted therapies for multiple myeloma, clinical challenges remain for patients with relapsed/refractory disease. A drug designed for efficient delivery of an alkylating payload into tumor cells that yields a favorable therapeutic window can be an attractive choice. Herein we describe melphalan flufenamide (melflufen), a drug with a peptide carrier component conjugated to an alkylating payload, and its cellular metabolism. We further underline the fundamental role of enzymatic hydrolysis in the rapid and robust accumulation of alkylating metabolites in cancer cells and their importance for downstream effects. The formed alkylating metabolites were shown to cause DNA damage, both on purified DNA and on chromatin in cells, with both nuclear and mitochondrial DNA affected in the latter. Furthermore, the rapid intracellular enrichment of alkylating metabolites is shown to be essential for the rapid kinetics of the downstream intracellular effects such as DNA damage signaling and induction of apoptosis. To evaluate the importance of enzymatic hydrolysis for melflufen's efficacy, all four stereoisomers of the compound were studied in a systematic approach and shown to have a different pattern of metabolism. In comparison with melflufen, stereoisomers lacking intracellular accumulation of alkylating payloads showed cytotoxic activity only at significantly higher concentration, slower DNA damage kinetics, and different mechanisms of action to reach cellular apoptosis.

Detoxification and removal of arsenite by Pseudomonas sp. SMS11: Oxidation, biosorption and bioaccumulation.

Arsenite [As(III)] oxidizing bacteria have been widely studied for their detoxification ability through transforming As(III) into arsenate [As(V)]. However, few was focused on removal capacity of arsenic (As). In the current study, As(III) oxidation accompanied with removal of total As was observed in Pseudomonas sp. SMS11. The biosorption (unbinding and surface binding) and bioaccumulation (intracellular uptake) of As by the cells were investigated. Biosorption isotherm was defined adequately by Langmuir and Freundlich models. Biosorption kinetics was recommended by pseudo second-order model. For comparison, the bacteria were inoculated in pure water or culture media amended with different concentrations of As(III) to evaluate the remediation capacity without or with bacterial growth. After removing unbound As, surface bound and intracellular As were sequentially separated using EDTA elution and acidic extraction from bacterial cells. Without bacterial growth, oxidation of As(III) was retarded and the maximum values of surface bound and intracellular As were 4.8 and 10.5 mg/g, respectively. Efficient oxidation and high adsorption capacity were observed after bacterial growth. The surface bound and intracellular As achieved up to 555.0 and 2421.5 mg/g, respectively. Strain SMS11 exhibited great accumulation capacity of As in aqueous solutions, indicating potential application in detoxification and removal of As(III) contamination. The results also suggested that bioremediation via bacteria should be based on living cells and bacterial growth rate.

In vitro toxicity and internalization of gold nanoparticles (AuNPs) in human epithelial colorectal adenocarcinoma (Caco-2) cells and the human skin keratinocyte (HaCaT) cells.

Ingestion and transdermal delivery are two common routes of nanoparticle (NP) exposure. In this study, the intracellular uptake, cytotoxicity and genotoxicity of 14 nm and 20 nm citrate-stabilized gold nanoparticles (AuNPs), 14 nm polyethylene glycol (PEG)-liganded carboxyl AuNPs, 14 nm PEG-liganded hydroxyl AuNPs and 14 nm PEG-liganded amine AuNPs were assessed on human epithelial colorectal adenocarcinoma (Caco-2) cells and the human skin keratinocyte (HaCaT) cells. The uptake of AuNPs in the cells was confirmed through darkfield microscopy and hyperspectral imaging followed by spectral angle mapping (SAM). A high level of citrate AuNPs was found in both cell lines whilst uptake of PEGylated AuNPs was low, irrespective of their functional groups. Cytotoxicity assessed by cell impedance was only observed for the 14 nm citrate-stabilized AuNPs. Enhanced cell proliferation was also observed in 14 nm PEG-liganded hydroxyl and 14 nm PEG-liganded amine AuNP-treated Caco-2 and HaCaT cells. For the assessment of genotoxicity, the in vitro micronucleus assay was used. Dose-dependent genotoxicity was observed in both Caco-2 and HaCaT cells, with all the AuNPs inducing genotoxicity. In conclusion, the entry of NPs into the cells as well as toxicity was dependent on their physicochemical properties such as surface coating and different chemical functional groups.

Different Influences of Biotinylation and PEGylation on Cationic and Anionic Proteins for Spheroid Penetration and Intracellular Uptake to Cancer Cells.

To better understand the effects of PEGylation and biotinylation on the delivery efficiency of proteins, the cationic protein lysozyme (LZ) and anionic protein bovine serum albumin (BSA) were chemically conjugated with poly(ethylene glycol) (PEG) and biotin-PEG to primary amine groups of proteins using N-hydroxysuccinimide reactions. Four types of protein conjugates were successfully prepared: PEGylated LZ (PEG-LZ), PEGylated BSA (PEG-BSA), biotin-PEG-conjugated LZ (Bio-PEG-LZ), and biotin-PEG-conjugated BSA (Bio-PEG-BSA). PEG-LZ and Bio-PEG-LZ exhibited a lower intracellular uptake than that of LZ in A549 human lung cancer cells (in a two-dimensional culture). However, Bio-PEG-BSA showed significantly improved intracellular delivery as compared to that of PEG-BSA and BSA, probably because of favorable interactions with cells via biotin receptors. For A549/fibroblast coculture spheroids, PEG-LZ and PEG-BSA exhibited significantly decreased tissue penetration as compared with that of unmodified proteins. However, Bio-PEG-BSA showed tissue penetration comparable to that of unmodified BSA. In addition, citraconlyated LZ (Cit-LZ) showed reduced spheroid penetration as compared to that of LZ, probably owing to a decrease in protein charge. Taken together, chemical conjugation of targeting ligands-PEG to anionic proteins could be a promising strategy to improve intracellular delivery and in vivo activity, whereas modifications of cationic proteins should be more delicately designed.

Nanoencapsulation of Ru(p-cymene) Complex Bearing Ginger-based Natural Product into Liposomal Nanoformulation to Improve Its Cellular Uptake and Antiproliferative Activity.

The organometallic compounds are prospective candidates in the row of developing metallochemotherapeutics with the aim of overcoming the limitations of platinum drugs. In order to explore the anticancer properties of organometallic compounds with the natural medicines, two Ru(II)-p-cymene complexes containing the natural products, viz., 6-gingerol (6G) and benzylated-6-gingerdione (B-6GD) have been synthesized and characterized well. The phenolic group of the Ru(6G) complex facilitates its higher cell-free antioxidant activity than its analogue complex. Also, the same complex shows higher cytotoxicity toward A549 lung and HeLa-S3 cervical cancer cells than the Ru(B-6GD) complex but lower cytotoxicity toward A2058 metastatic melanoma cancer cells. Both complexes are shown to easily accumulate in melanoma cancer cells, and their degree of cytotoxicity in the same cells is found to be positively correlated with cell uptake. The cytotoxicity of complexes arises from their intracellular activity, mainly due to the induction of singlet oxygen production in cancer cells. The subcellular fractionation study shows that mitochondria and ER-Golgi membranes might be their predominant targets. Also, the mechanistic investigation revealed that Ru(B-6GD) induces caspase-dependent non-apoptotic cell death whereas Ru(6G) can induce caspase-independent non-apoptotic cell death. Furthermore, both complexes are found to moderately alter the adhesion properties of cancer cells, which is beneficial for antimetastatic treatment. Despite the potential pharmacological activity, Ru(6G) is encapsulated into polymer-supported liposomes to reduce its toxicity and further improve its anticancer potency. The π-conjugated yne-ene chain of polydiacetylene aids in the development of a stable nanoformulation, which achieved a slow release of the complex. Most importantly, the cancer cell uptake of the liposome-encapsulated Ru(6G) complex is 20 times enhanced and the total ROS formation in cancer cells is significantly increased compared to the non-encapsulated complex. However, the nanoformulation does not alter the antimetastatic potency of the encapsulated complex.

Luminescent Citrate-Functionalized Terbium-Substituted Carbonated Apatite Nanomaterials: Structural Aspects, Sensitized Luminescence, Cytocompatibility, and Cell Uptake Imaging.

This work explores the preparation of luminescent and biomimetic Tb3+-doped citrate-functionalized carbonated apatite nanoparticles. These nanoparticles were synthesized employing a citrate-based thermal decomplexing precipitation method, testing a nominal Tb3+ doping concentration between 0.001 M to 0.020 M, and a maturation time from 4 h to 7 days. This approach allowed to prepare apatite nanoparticles as a single hydroxyapatite phase when the used Tb3+ concentrations were (i) ≤ 0.005 M at all maturation times or (ii) = 0.010 M with 4 h of maturation. At higher Tb3+ concentrations, amorphous TbPO4·nH2O formed at short maturation times, while materials consisting of a mixture of carbonated apatite prisms, TbPO4·H2O (rhabdophane) nanocrystals, and an amorphous phase formed at longer times. The Tb3+ content of the samples reached a maximum of 21.71 wt%. The relative luminescence intensity revealed an almost linear dependence with Tb3+ up to a maximum of 850 units. Neither pH, nor ionic strength, nor temperature significantly affected the luminescence properties. All precipitates were cytocompatible against A375, MCF7, and HeLa carcinogenic cells, and also against healthy fibroblast cells. Moreover, the luminescence properties of these nanoparticles allowed to visualize their intracellular cytoplasmic uptake at 12 h of treatment through flow cytometry and fluorescence confocal microscopy (green fluorescence) when incubated with A375 cells. This demonstrates for the first time the potential of these materials as nanophosphors for living cell imaging compatible with flow cytometry and fluorescence confocal microscopy without the need to introduce an additional fluorescence dye. Overall, our results demonstrated that Tb3+-doped citrate-functionalized apatite nanoparticles are excellent candidates for bioimaging applications.

Doxorubicin Loaded Dextran-coated Superparamagnetic Iron Oxide Na-noparticles with Sustained Release Property: Intracellular Uptake, Phar-macokinetics and Biodistribution Study.

BACKGROUND: Due to the short biological half-life and serious side effects (especially for heart and kidney), the application of Doxorubicin (Dox) in clinical therapy is strictly limited. To overcome these shortcomings, a novel sustained release formulation of doxorubicin-loaded dextran-coated superparamagnetic iron oxide nanoparticles (Dox-DSPIONs) was prepared. OBJECTIVE: The purpose of this study was to evaluate the intracellular uptake behavior of Dox-DSPIONs and to investigate their pharmacokinetics and biodistribution properties. METHOD: Confocal laser scanning microscopy was employed to study the intracellular uptake and release properties of Dox from Dox-DSPIONs in SMMC-7721 cells. Simple high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method was established to study the pharmacokinetics and biodistribution properties of Dox-DSPIONs in vivo after intravenous administration and compared with free Dox. RESULTS: Intracellular uptake experiment indicated that Dox could be released sustainedly from Dox-DSPIONs over time. The pharmacokinetics parameters displayed that the T1/2and AUC0-24h of Dox-DSPIONs were higher than those of free Dox, while the Cmax of Dox-DSPIONs was significantly lower than that of free drug. The biodistribution behaviors of the drug were altered by Dox-DSPIONs in mice, which showed obvious liver targeting, and significantly reduced the distribution of the drug in the heart and kidney. CONCLUSION: Dox-DSPIONs have the sustained-release property in vitro and in vivo, which could significantly prolong blood circulation time, improve bioavailability, and reduce the side effects of Dox. Therefore, the novel formulation of the Dox-DSPIONs has the potential as a promising drug delivery system in cancer therapy.

Silmitasertib-induced macropinocytosis promoting DDP intracellular uptake to enhance cell apoptosis in oral squamous cell carcinoma.

Cisplatin (DDP) is a first-line chemotherapeutic drug applied for the treatment of oral squamous cell carcinoma (OSCC). The anticancer activity of DDP is tightly linked to its intracellular uptake. It is unwise to increase the DDP intake by increasing the dose or shortening the dosing interval because of the severe systemic toxicity (nephrotoxicity, ototoxicity and neurotoxicity) in DDP application. The main uptake pathways of DDP include passive diffusion and active transporter transport. Therefore, finding additional uptake pathways that can improve the effective intracellular concentration of DDP is critical. Macropinocytosis, an endocytic mechanism for extracellular material absorption, contributes to the intracellular uptake of anticancer drugs. No research has been conducted to determine whether macropinocytosis can augment the intracellular uptake of DDP in OSCC cells or not. Based on that, we proved for the first time that silmitasertib (previously CX-4945) could trigger macropinocytosis, which may increase the intracellular uptake of DDP and enhance apoptosis via in vivo and in vitro experiments. We hope that our findings will inspire a new approach for the application of DDP in cancer treatment.

Weakly acidic carboxy group-grafted ß-cyclodextrin-threaded acid-degradable polyrotaxanes for modulating protein interaction and cellular internalization.

To improve the therapeutic potential of ß-cyclodextrin (ß-CD)-threaded acid-degradable polyrotaxanes (ß-CD PRXs) in cholesterol-related metabolic disorders, we investigated the effect of carboxylation of ß-CD PRXs on intracellular uptake. In this study, we established a synthetic method for the modification of carboxylalkyl carbamates on ß-CD PRXs without degradation and synthesized three series of carboxyalkyl carbamate group-modified ß-CD PRXs with different alkyl spacer lengths. The modification of carboxymethyl carbamate (CMC), carboxyethyl carbamate (CEC), and carboxypropyl carbamate (CPC) on the ß-CD PRXs slightly reduced the interaction of the PRXs with the lipid layer model compared with the modification of 2-(2-hydroxyethoxy)ethyl carbamate (HEE-PRX), which was used in our previous studies. However, all the carboxylated ß-CD PRXs showed a significantly stronger interaction with a protein model compared with HEE-PRX. The carboxylated ß-CD PRXs showed significantly high intracellular uptake, through macrophage scavenger receptor A (MSR-A)-mediated endocytosis, in MSR-A-positive RAW 264.7 cells compared with HEE-PRX. Interestingly, the carboxylated ß-CD PRXs also showed significantly higher intracellular uptake even in MSR-A-negative cells compared with HEE-PRX. Carboxylated ß-CD PRXs are considered to strongly interact with other membrane proteins, resulting in high intracellular uptake. The length of the alkyl spacer affected the intracellular uptake levels of carboxylated PRXs, however, this relationship was varied for different cell types. Furthermore, none of the carboxylated ß-CD PRXs exhibited cytotoxicity in the RAW 264.7 and NIH/3T3 cells. Altogether, carboxylation of ß-CD PRXs is a promising chemical modification approach for their therapeutic application because carboxylated ß-CD PRXs exhibit high cellular internalization efficiency in MSR-A-negative cells and negligible toxicity.

New Insight into the Fluorescence Quenching of Nitrogen-Containing Carbonaceous Quantum Dots-From Surface Chemistry to Biomedical Applications.

Carbon-based quantum dots are widely suggested as fluorescent carriers of drugs, genes or other bioactive molecules. In this work, we thoroughly examine the easy-to-obtain, biocompatible, nitrogen-containing carbonaceous quantum dots (N-CQDs) with stable fluorescent properties that are resistant to wide-range pH changes. Moreover, we explain the mechanism of fluorescence quenching at extreme pH conditions. Our in vitro results indicate that N-CQDs penetrate the cell membrane; however, fluorescence intensity measured inside the cells was lower than expected from carbonaceous dots extracellular concentration decrease. We studied the mechanism of quenching and identified reduced form of ß-nicotinamide adenine dinucleotide (NADH) as one of the intracellular quenchers. We proved it experimentally that the elucidated redox process triggers the efficient reduction of amide functionalities to non-fluorescent amines on carbonaceous dots surface. We determined the 5 nm-wide reactive redox zone around the N-CQD surface. The better understanding of fluorescence quenching will help to accurately quantify and dose the internalized carbonaceous quantum dots for biomedical applications.

Intracellular uptake of nanocrystals: Probing with aggregation-induced emission of fluorescence and kinetic modeling.

Nanocrystal formulations have been explored to deliver poorly water-soluble drug molecules. Despite various studies of nanocrystal formulation and delivery, much more understanding needs to be gained into absorption mechanisms and kinetics of drug nanocrystals at various levels, ranging from cells to tissues and to the whole body. In this study, nanocrystals of tetrakis (4-hydroxyphenyl) ethylene (THPE) with an aggregation-induced emission (AIE) property was used as a model to explore intracellular absorption mechanism and dissolution kinetics of nanocrystals. Cellular uptake studies were conducted with KB cells and characterized by confocal microscopy, flow cytometry, and quantitative analyses. The results suggested that THPE nanocrystals could be taken up by KB cells directly, as well as in the form of dissolved molecules. The cellular uptake was found to be concentration- and time-dependent. In addition, the intracellular THPE also could be exocytosed from cells in forms of dissolved molecules and nanocrystals. Kinetic modeling was conducted to further understand the cellular mechanism of THPE nanocrystals based on first-order ordinary differential equations (ODEs). By fitting the kinetic model against experimental measurements, it was found that the initial nanocrystal concentration had a great influence on the dynamic process of dissolution, cellular uptake, and exocytosis of THPE nanocrystals. As the nanocrystal concentration increased in the culture media, dissolution of endocytosed nanocrystals became enhanced, subsequently driving the efflux of THPE molecules from cells.

Streptomycin sulphate loaded solid lipid nanoparticles show enhanced uptake in macrophage, lower MIC in Mycobacterium and improved oral bioavailability.

Present study addresses the challenge of incorporating hydrophilic streptomycin sulphate (STRS; log P -6.4) with high dose (1 g/day) into a lipid matrix of SLNs. Cold high-pressure homogenization technique used for SLN preparation achieved 30% drug loading and 51.17 ± 0.95% entrapment efficiency. Polyethylene glycol 600 as a supporting-surfactant assigned small size (218.1 ± 15.46 nm) and mucus-penetrating property. It was conceived to administer STRS-SLNs orally rather than intramuscularly. STRS-SLNs remained stable on incubation for varying times in SGF or SIF. STRS-SLNs were extensively characterised for microscopic (TEM and AFM), thermal (DSC), diffraction (XRD) and spectroscopic (NMR and FTIR) properties and showed zero-order controlled release. Enhanced (60 times) intracellular uptake was observed in THP-1 and Pgp expressing LoVo and DLD-1 cell lines, using fluorescein-SLNs. Presence of SLNs in LoVo cells was also revealed by TEM studies. STRS-SLNs showed 3 times reduction in MIC against Mycobacterium tuberculosis H37RV (256182) in comparison to free STRS. It also showed better activity against both M. bovis BCG and Mycobacterium tuberculosis H37RV (272994) in comparison to free STRS. Cytotoxicity and acute toxicity studies (OECD 425 guidelines) confirmed in vitro and in vivo safety of STRS-SLNs. Single-dose oral pharmacokinetic studies in rat plasma using validated LCMS/MS technique or the microbioassay showed significant oral absorption and bioavailability (160% - 710% increase than free drug).

Distinct Proteins in Protein Corona of Nanoparticles Represent a Promising Venue for Endogenous Targeting - Part I: In vitro Release and Intracellular Uptake Perspective.

INTRODUCTION: Protein corona (PC) deposition on nanoparticles (NPs) in biological systems contributes to a great extent to NPs' fates; their targeting potential, the interaction with different biological systems and the subsequent functions. PC - when properly tuned - can serve as a potential avenue for optimization of NPs' use in cancer therapy. METHODS: Poly-lactic co-glycolic acid (PLGA)-based NPs exhibiting different physicochemical properties were fabricated and characterized. The PC makeup of these NPs were qualitatively and quantitatively analyzed by Western blot and Bradford assay, respectively. The effect of PC on the release of NPs' cargos and the intracellular uptake into B16F10 melanoma cells has been studied. RESULTS: The composition of NPs (polymeric PLGA NPs vs lipid-polymer hybrid NPs) and the conjugation of an active targeting ligand (cRGDyk peptide) represented the major determinants of the PC makeup of NPs. The in vitro release of the loaded cargos from the NPs depended on the PC and the presence of serum proteins in the release medium. Higher cumulative release has been recorded in the presence of proteins in the case of peptide conjugated NPs, cNPs, while the unconjugated formulations, uNPs, showed an opposite pattern. NPs intracellular uptake studies revealed important roles of distinct serum and cellular proteins on the extent of NPs' accumulation in melanoma cells. For example, the abundance of vitronectin (VN) protein from serum has been positively related to the intracellular accumulation of the NPs. CONCLUSION: Careful engineering of nanocarriers can modulate the recruitment of some proteins suggesting a potential use for achieving endogenous targeting to overcome the current limitations of targeted delivery of chemotherapeutic agents.

Formulation and evaluation of hyaluronic acid-based mucoadhesive self nanoemulsifying drug delivery system (SNEDDS) of tamoxifen for targeting breast cancer.

The present study was intended to develop a papain grafted S-protected hyaluronic acid-lithocholic acid co-block (PAP-HA-ss-LCA) polymeric excipient as an amphiphilic muco permeating stabilizer for targeting breast cancer epithelial cells overexpressed with CD44 receptors. The mucopermeating, stabilizing and targeting capability of the PAP-HA-ss-LCA polymeric excipient was investigated by manufacturing tamoxifen (TMX) loaded self-nanoemulsifying drug delivery system (SNEDDS). TMX loaded PAP-HA-ss-LCA incorporated SNEDDS (TMX-PAP-HA-ss-LCA SNEDDS) were characterized for their surface chemistry, drug release, permeation enhancement, biocompatibility and antitumor activity. FTIR spectroscopic analysis showed successful synthesis of PAP-HA-ss-LCA polymer. X-ray diffraction (XRD) showed the amorphous form of TMX inside SNEDDS. The observed hydrodynamic diameter of TMX-PAP-HA-ss-LCA SNEDDS was 367.5 nm. Furthermore, Hyaluronic Acid-based Mucoadhesive Self Nanoemulsifying Drug Delivery System (SNEDDS) of TMX showed homogeneity in synthesis with low polydispersity and negative zeta potential due to stabilization with PAP-HA-ss-LCA polymer. The distinct spherical shape of the nanodroplets was evident by transmission electron microscopy (TEM). In vitro release kinetics indicated approximately >80% release within 48 h under sink conditions. Ex-vivo permeation study displayed 7.11-folds higher permeation of TMX by TMX-PAP-HA-ss-LCA in contrast to pure TMX. The biocompatibility study proved that SNEDDS formulation was safe and compatible against macrophages. In vitro cytotoxicity studies demonstrated that TMX-PAP-HA-ss-LCA SNEDDS could efficiently kill MCF-7 breast cancer cells as compared to the native TMX drug. Systemic toxicity studies proved the non-toxic nature of TMX-PAP-HA-ss-LCA in contrast to pure TMX. Based on these evidences, TMX-PAP-HA-ss-LCA SNEDDS formulation seems to be promising mucopermeating, augmented intracellular uptake with strong targeting potential for anti-proliferative activity.