Preparation, Characterization and In vitro Evaluation of Insulin-PHBV Nanoparticles / Alginate Hydrogel Composite System for Prolonged Delivery of Insulin.

PURPOSE: In the present study, biodegradable poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanoparticles (NPs) containing insulin were loaded in sodium alginate/jeffamine (ALG/jeff) hydrogel for prolonged delivery of insulin. The main aim of this work was to fabricate an efficient insulin delivery system to improve patient adherence by decreasing the repetition of injections. METHODS: Swelling and morphological properties and crosslinking efficiency of ALG/jeff hydrogel were assessed. The composite hydrogel was prepared by adding PHBV NPs to ALG/jeff hydrogel concurrently with crosslinking process. The morphology and loading capacity of composite hydrogel were analyzed. RESULTS: Circular dichroism measurement demonstrated that insulin remains stable following fabrication process. The release profile exhibited 54.6 % insulin release from composite hydrogel within 31 days with minor initial burst release equated to nanoparticles and hydrogels. MTT cell viability analysis was performed by applying L-929 cell line and no cytotoxic effect was observed. CONCLUSIONS: Favorable results clearly introduced fabricated composite hydrogel as an excellent candidate for drug delivery systems and also paves the route for prolonged delivery systems of other proteins.

Review of recent preclinical and clinical research on ligand-targeted liposomes as delivery systems in triple negative breast cancer therapy.

Triple-negative breast Cancer (TNBC) is one of the deadliest types, making up about 20% of all breast cancers. Chemotherapy is the traditional manner of progressed TNBC treatment; however, it has a short-term result with a high reversibility pace. The lack of targeted treatment limited and person-dependent treatment options for those suffering from TNBC cautions to be the worst type of cancer among breast cancer patients. Consequently, appropriate treatment for this disease is considered a major clinical challenge. Therefore, various treatment methods have been developed to treat TNBC, among which chemotherapy is the most common and well-known approach recently studied. Although effective methods are chemotherapies, they are often accompanied by critical limitations, especially the lack of specific functionality. These methods lead to systematic toxicity and, ultimately, the expansion of multidrug-resistant (MDR) cancer cells. Therefore, finding novel and efficient techniques to enhance the targeting of TNBC treatment is an essential requirement. Liposomes have demonstrated that they are an effective method for drug delivery; however, among a large number of liposome-based drug delivery systems annually developed, a small number have just received authorization for clinical application. The new approaches to using liposomes target their structure with various ligands to increase therapeutic efficiency and diminish undesired side effects on various body tissues. The current study describes the most recent strategies and research associated with functionalizing the liposomes' structure with different ligands as targeted drug carriers in treating TNBCs in preclinical and clinical stages.

Targeted Delivery of Anticancer Drug Loaded Charged PLGA Polymeric Nanoparticles Using Electrostatic Field.

Pure positive electrostatic charges (PPECs) show suppressive effect on the proliferation and metabolism of invasive cancer cells without affecting normal tissues. PPECs are used for the delivery of drug-loaded polymeric nanoparticles (DLNs) capped with negatively charged poly(lactide-co-glycolide) (PLGA) and Poly(vinyl-alcohol) PVA into the tumor site of mouse models. The charged patch is installed on top of the skin in the mouse models' tumor region, and the controlled selective release of the drug is assayed by biochemical, radiological, and histological experiments on both tumorized models and normal rats' livers. It is found that DLNs synthesized by PLGA show great attraction to PPECs due to their stable negative charges, which would not degrade immediately in blood. The burst and drug release after less than 48h of this synthesized DLNs are 10% and 50%, respectively. These compounds can deliver the loaded-drug into the tumor site with the assistance of PPECs, and the targeted-retarded release will take place. Hence, local therapy can be achieved with much lower drug concentration (conventional chemotherapy [2 mg kg-1 ] versus DLNs-based chemotherapy [0.75 mg kg-1 ]) with negligible side effects in non-targeted organs. PPECs have many potential clinical applications for advanced-targeted chemotherapy with the lowest discernible side effects.

Co-delivery systems: hope for clinical application?

Cancer is a multidimensional and challenging disease to handle. Current statistics reveal that we are far from satisfying cancer treatment. Taking advantage of different therapeutic agents that affect multiple pathways has been established as highly productive. Nevertheless, owing to several hindrances to conventional combination therapy, such as lack of tumor targeting, non-uniform pharmacokinetic of the combined drugs, and off-target side effects, it is well documented that this treatment approach is unlikely to address all the difficulties observed in monotherapy. Co-delivery systems could enhance the therapeutic efficacy of the combination therapy by targeting cancer cells and improving the pharmacokinetic and physicochemical properties of the therapeutic agents. Nevertheless, it seems that present knowledge in responding to the challenges in cancer treatment is still inadequate and far from optimal treatment, which highlights the urgent need for systematic studies direct to identify various aspects of co-delivery systems. Accordingly, to gather informative data, save time, and achieve superior results, the following steps are necessary: (1) implementing computational methods to predict drug-drug interactions (DDIs) in vitro and in vivo, (2) meticulous cancer studies at the cellular and molecular levels to obtain specific criteria for selecting preclinical and clinical models, (3) extensive physiological and pharmacokinetic study of nanocarriers behavior in preclinical models, and (4) finding the optimal formulation and analyzing its behavior in cellular and animal models facilitates bridging in vivo models to clinical trials. This review aims to deliver an overview of co-delivery systems, rationales, and suggestions for further studies in this field.

Effect of oral administration of GnRHa+nanoparticles of chitosan in oogenesis acceleration of goldfish Carassius auratus.

Several methods have been used to accelerate previtellogenesis and vitellogenesis stages in fish, including hormonal induction, sustained-release delivery systems, and oral delivery of gonadotropin-releasing hormone (GnRH). In this study, we proposed the oral administration of GnRH analog + nanoparticles of chitosan to accelerate oogenesis in goldfish as a model fish in reproductive biology and aquaculture. In this regard, adult female goldfish were fed with six experimental groups: chitosan, 50 µg GnRHa/kg b.w., 100 µg GnRHa/kg b.w., chitosan + 50 µg GnRHa/kg b.w., and chitosan + 100 µg GnRHa/kg b.w., and diet without any additive as the control for 40 days in triplicate. Every 10 days, ovarian samples were collected, and gonadosomatic index (GSI), oocyte diameter (OD), zona radiata thickness (Zr), and diameter of the follicular layer (Fl) were measured to assess ovarian developmental stage for each treatment. Additionally, blood sampling was done to measure serum 17ß-estradiol concentration at the end of the experiment. All parameters remained unchanged during the experiment in the chitosan-fed group. In the group fed with 100 µg GnRH or chitosan nanoparticle + 100 µg GnRHa, these parameters in general were increased. However, the effects in 50 µg GnRHa or chitosan nanoparticle + 50 µg GnRHa treatments were uncertain; they affected serum E2 levels as a trend toward a significant increase was observed in goldfish treated with chitosan nanoparticle + 100 µg GnRHa. Finally, the results indicated the oral administration of chitosan + 100 µg GnRHa/kg b.w. significantly accelerated the oocyte development and growth of ovary.

Chloroquine Assisted Delivery of microRNA Mimic Let-7b to NSCLC Cell Line by PAMAM (G5) - HA Nano-Carrier.

AIM: Polyamidoamine (PAMAM) dendrimers are attracting interest of the scientists as vehicles for nucleic acid delivery due to their suitable properties. The highly positive surface charged of PAMAM enables an adequate interaction with negatively charged microRNAs. PURPOSE: The purpose of this study is to investigate the anti-tumor effect of microRNA Mimic let-7b loaded in PAMAM dendrimers (G5) on Non-Small Cell Lung Cancer (NSCLC) cells. OBJECTIVE: In order to increase the anti-tumor effect, chloroquine is employed to enhance the endosomal escape which is counted as a limitation in the advancement of gene delivery. Nanoparticles (NPs) were coated with natural polysaccharide "Hyaluronic Acid (HA)" to develop biodegradable carriers with targeting moiety for over-expressed CD44 receptors on NSCLC cells. The size and zeta potential measurements, gel retardation, cellular uptake, cell viability and gene expression studies were investigated for the designed delivery system. RESULTS: DLS analysis showed monodispersed small nanoparticles, which was in agreement with TEM results. Remarkably, NPs in the cells pretreated with chloroquine exhibited the highest cytotoxicity and were capable of inducing apoptosis. In cellular uptake study, NPs labeled with Fluorescein Isothiocyanate (FITC), were successfully taken up in cancer cells. Moreover, the expression study of three genes linked with cancer initiation and development in NSCLC, including KRAS, p-21, and BCL-2 indicated a decrease in KRAS and BCL-2 (oncogenic and anti-apoptotic genes) and increase in p-21 (apoptotic gene). CONCLUSION: All factors considered, the results declare that application of let-7b-loaded PAMAM-HA NPs in combination with chloroquine can be a promising therapeutic option in cancer cells inhibition. This fact has frequently been highlighted by many researchers upon the potentials of micro RNA delivery in cancer cells.

Temperature-Responsive Methylcellulose-Hyaluronic Hydrogel as a 3D Cell Culture Matrix.

This study investigated the application of a temperature-responsive methylcellulose-hyaluronic acid (MC-HA) hydrogel to support 3D cell growth in vitro. Initial work focused on the preparation of hydrogels for 3D culture, followed by investigations of the biological compatibility of hydrogel components and optimization of the cell culture environment. Evaluation of viability and proliferation of HCT116 cells cultured in the MC-HA hydrogel was used to adjust the blend composition to design a hydrogel with optimal properties to support cell growth. Two important aspects in terms of the application of the proposed polymeric matrix in 3D cell culture were demonstrated: (i) 3D cultured cell aggregates can be released/recovered from the matrix via a gentle procedure that will preserve cell viability and (ii) the hydrogel matrix is amenable to application in a 96-well plate format as a standard approach employed in in vitro tissue culture tests. The work therefore shows that MC-HA hydrogels demonstrate potential for in vitro 3D cell culture as inexpensive and well-defined alternatives to animal-derived or complex synthetic systems.

Mitochondrial delivery of microRNA mimic let-7b to NSCLC cells by PAMAM-based nanoparticles.

Many biological mechanisms including cellular metabolism and cell death are regulated by mitochondria known as powerhouse of the cell. Recently, let-7b, a tumour-suppressor microRNA has been detected in mitochondria of human cells targeting several mitochondrial-encoded respiratory chain genes. Triphenylphosphonium cation (TPP) is one of the major classes of mitochondriotropics that possess the ability of specifically targeting the mitochondria. PAMAM dendrimers are one of the most available agents in gene delivery due to their well-defined and beneficial features such as large density of surface functional groups. Hyaluronic acid (HA), a natural polysaccharide has been demonstrated to have the abilities such as good biocompatibility and targeting CD44 overexpressed receptors on non-small cell lung cancer (NSCLC) cells. In this research, let-7b-PAMAM (G5)-TPP and let-7b-PAMAM (G5)-TPP-HA nano-carriers were designed to deliver let-7b miRNA mimic to NSCLC cells' mitochondria as a novel way of cancer cells inhibition. Nano-carriers were capable of being successfully taken up by A549 cells and localised in mitochondria environment. Let-7b loaded nanoparticles reduced cell viability and induced apoptosis significantly. Expression of genes involved in mitochondrial oxidative function was decreased resulting in nanoparticles effect on mitochondria. Application of mitochondria targeted-miRNA delivery systems could regulate cellular functions to inhibit lung cancer.

Improving the in-vivo biological activity of fingolimod loaded PHBV nanoparticles by using hydrophobically modified alginate.

Uncontrolled distribution of nanoparticles (NPs) within the body can significantly decrease the efficiency of drug therapy and is considered among the main restrictions of NPs application. The aim of this study was to develop a depot combination delivery system (CDS) containing fingolimod loaded poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) NPs dispersed into a matrix of oleic acid-grafted-aminated alginate (OA-g-AAlg) to minimize the nonspecific biodistribution (BD) of PHBV NPs. OA-g-AAlg was synthesized in two step; First, Alg was aminated by using adipic dihydrazide (ADH). The degree of hyrazide group substitution of Alg was determined by trinitro-benzene-sulfonic acid (TNBS) assay. Second, OA was attached to AAlg through formation of an amide bond. Chemical structure of OA-g-AAlg was confirmed with FTIR and HNMR spectroscopy. Furthermore, rheological properties of OA-g-AAlg with different grafting ratios were evaluated. In-vitro release studies indicated that 47% of fingolimod was released from the CDS within 28 days. Blood and tissue samples were analyzed using liquid chromatography/tandem mass spectrometry following subcutaneous (SC) injection of fingolimod-CDS into Wistar rats. The elimination phase half-life of CDS-fingolimod was significantly higher than that of fingolimod (∼32 d vs. ∼20 h). To investigate the therapeutic efficacy, lymphocyte count was assessed over a 40 day period in Wistar rats. Peripheral blood lymphocyte count decreased from baseline by 27 ± 8% in 2 days after injection. Overall, the designed CDS represented promising results in improving the pharmacokinetic properties of fingolimod. Therefore, we believe that this sustained release formulation has a great potential to be applied to delivery of various therapeutics.

ADCs, as Novel Revolutionary Weapons for Providing a Step Forward in Targeted Therapy of Malignancies.

Antibody drug conjugates (ADCs), as potent pharmaceutical trojan horses for cancer treatment, provide superior efficacy and specific targeting along with low risk of adverse reactions compared to traditional chemotherapeutics. In fact, the development of these agents combines the selective targeting capability of monoclonal antibody (mAb) with high cytotoxicity of chemotherapeutics for controlling the neoplastic mass growth. Different ADCs (more than 60 ADCs) in preclinical and clinical trials were introduced in this novel pharmaceutical field. Various design-based factors must be taken into account for improving the functionality of ADC technology, including selection of appropriate target antigen and high binding affinity of fragment (miniaturized ADCs) or full mAbs (preferentially use of humanized or fully human antibodies compared to murine and chimeric ones), use of bispecific antibodies for dual targeting effect, linker engineering and conjugation method efficacy to obtain more controlled drug to antibody ratio (DAR). Challenging issues affecting therapeutic efficacy and safety of ADCs, including bystander effect, on- and off-target toxicities, multi drug resistance (MDR) are also addressed. 4 FDA-approved ADCs in the market, including ADCETRIS ®, MYLOTARG®, BESPONSA ®, KADCYLA®. The goal of the current review is to evaluate the key parameters affecting ADCs development.

PHBV/PLGA nanoparticles for enhanced delivery of 5-fluorouracil as promising treatment of colon cancer.

5-Fluorouracil (5-FU) is one of the most widely used agents in the first-line chemotherapy for colon cancer. However, clinical use of 5-FU is limited because of the low efficacy of drug uptake and systemic toxic effects. Therefore, there is a critical need to find better drug delivery systems in order to improve the efficacy of the drug. In the present study, we have developed a novel combination drug delivery system based on PHBV/PLGA NPs for delivery of 5-FU to cancer cells. NPs were prepared by the double emulsion method and their optimization of preparation was evaluated using Box-Behnken design (BBD) of response surface methodology (RSM). 5-FU loaded NPs were characterized by scanning electron microscope (SEM), differential scanning calorimetry (DSC), thermogravimetry analysis (TGA), and Fourier transformed infra-red spectroscopy (FT-IR). SEM image implied that NPs were spherical in shape and the results of DSC, TGA, and FT-IR suggest that 5-FU was encapsulated into NPs. The obtained results revealed that 5-FU loaded PHBV/PLGA NPs induced significant higher cell death at concentration much lower than free 5-FU. Results of hemolysis assay indicated that the NPs were hemo-compatible. In vivo anti-tumor studies showed that 5-FU loaded NPs reduced tumor volume significantly in comparison with free 5-FU. As the first example of using PHBV/PLGA as nano-drug delivery system with enhanced anti-tumor activities, this study establishes PHBV/PLGA as a novel promising drug delivery platform for treatment of colon cancer.

New folate receptor targeted nano liposomes for delivery of 5-fluorouracil to cancer cells: Strong implication for enhanced potency and safety.

We previously showed that folate liposomes of 5FU made from Dipalmitoylphosphatidylcholine (DPPC) induced cell death in HT-29 and HeLa cells more potently than bulk 5FU. Also, a primary 5FU liposomal formulation with phosphatidyl choline (PC) exhibited higher cytotoxicity in murine colon cancer cells. In the present study, optimization of 5FU PC liposome, mechanism of cell death induction in human cancer cell lines and its safety along with other assays have been employed for targeted PC liposomes of 5FU. Liposomes were prepared using thin layer method and optimization of preparation was assessed using central composite design (CCD) of response surface methodology (RSM). Folic acid (FA) was employed as the targeting ligand. Morphology of 5FU loaded liposomes and changes in their thermal behavior were assessed by transmission electron microscopy (TEM) and differential scanning calorimetry (DSC), respectively. In vitro cytotoxicity was explored using MTT assay in HT-29, Caco-2, HeLa and MCF-7 cell lines. Cytotoxicity mechanism of the targeted delivery system was searched through the evaluation of reactive oxygen species (ROS) overproduction, mitochondrial membrane potential (∆Ψm), the release of cytochrome c, the activity of caspase 3/7 and apoptosis and necrosis rate. Liposomes were spherical in shape and 5FU was successfully encapsulated into liposomes rather in an amorphous state. Our interesting results showed that in HT-29 cells targeted liposomes triggered the mitochondrial apoptotic pathway by decreasing the mitochondrial membrane potential, releasing of cytochrome c and promoting the substantial activity of caspase 3/7. In HeLa cells, however, targeted liposomes particularly activated necrosis pathway through the overproduction of ROS. Folate-liposomal 5FU showed significantly higher antitumor efficiency compared to free drug. The results of this study offer new prospects for cancer therapy with reducing systemic drug exposure and associated toxicities.

Co-Delivery Nanosystems for Cancer Treatment: A Review.

Massive data available on cancer therapy more than ever lead our mind to the general concept that there is no perfect treatment for cancer. Indeed, the biological complexity of this disease is too excessive to be treated by a single therapeutic approach. Current delivery systems containing a specific drug or gene have their particular opportunities and restrictions. It is worth noting that a considerable number of studies suggest that single- drug delivery systems result in insufficient suppression of cancer growth. Therefore, one of the main ideas of co-delivery system designing is to enhance the intended response or to achieve the synergistic/combined effect compared to the single drug strategy. This review focuses on various strategies for co-delivery of therapeutic agents in the treatment of cancer. The primary approaches within the script are categorized into co-delivery of conventional chemotherapeutics, gene-based molecules, and plant-derived materials. Each one is explained in examples with the recent researches. In the end, a brief summary is provided to conclude the gist of the review.

Fabrication of long-acting insulin formulation based on poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanoparticles: preparation, optimization, characterization, and in vitro evaluation.

The purpose of this research was the fabrication, statistical optimization, and in vitro characterization of insulin-loaded poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanoparticles (INS-PHBV-NPs). Nanopar-ticles were successfully developed by double emulsification solvent evaporation method. The NPs were characterized for particle size, entrapment efficiency (EE%), and polydispersity index (PDI). The NPs also were characterized by scanning electron microscopy (SEM), Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and circular dichroism (CD). The optimum conditions were found to be 1.6% polyvinyl alcohol (PVA), 0.9% of PHBV, and 15 mg/ml of insulin with the aid of the Box-Behnken experimental design results. The optimized NPs showed spherical shape with particle size of 250.21 ± 11.37 nm, PDI of 0.12 ± 0.01, and with EE% of 90.12 ± 2.10%. In vitro drug release pattern followed Korsmeyer-Peppas model and exhibited an initial burst release of 19% with extended drug release of 63.2% from optimized NPs within 27 d. In conclusion, these results suggest that INS-PHBV-NPs could be a promising candidate for designing an injectable sustained release formulation for insulin.

Comparison of 0.2% chlorhexidine mouthwash with and without anti-discoloration system in patients with chronic periodontitis: A randomized controlled clinical trial.

Background: Chemical plaque control, an adjunct to mechanical approaches, could improve the maintenance of patients with different types of periodontitis. Chlorhexidine, the gold standard in chemical plaque control, might have some side effects; the most determining one is tooth discoloration. Anti-discoloration systems (ADS) have been added to minimize brownish tooth discoloration. This study aimed to evaluate the staining potential and clinical efficacy of chlorhexidine with and without ADS in patients with chronic periodontitis. Methods: In this randomized controlled trial, 46 patients with chronic periodontitis were randomly allocated to two groups. Each patient used 10 mL of mouthwash A (CHX without ADS) or B (CHX with ADS, including sodium metabisulfite and ascorbic acid) twice a day for two weeks. After a two-week interval, they used the second mouthwash. At the beginning and the end of each two-week cycle, plaque index (PI), bleeding on probing (BoP), and staining index were recorded. Results: There was no significant difference between mouthwash A and B in the reduction of BoP and PI. The staining index was significantly lower after rinsing with mouthwash B compared to mouthwash A. Conclusion: CHX mouthwash containing ADS has similar efficacy in microbial plaque control and reduction of BOP as CHX without ADS, with the advantage of lower stain formation on tooth surfaces in patients with chronic periodontitis.

Co-delivery of 5-fluorouracil and oxaliplatin in novel poly(3-hydroxybutyrate-co-3-hydroxyvalerate acid)/poly(lactic-co-glycolic acid) nanoparticles for colon cancer therapy.

In the present study, a novel 5FU and OXA co-loaded PHBV/PLGA NPs was developed which induced apoptosis in cancer cells. NPs were prepared by the double emulsion method and their preparation was optimized using D-optimal design of response surface methodology (RSM). 5FU-OXA loaded NPs were evaluated by SEM, DSC and DLS. NPs were spherical as shown by SEM and the results of DSC indicated that both drugs successfully entrapped into NPs. 5FU-OXA loaded NPs exhibited higher cytotoxicity effect than free drugs on cancer cells. For the first time to our knowledge, these results showed that more ROS generation and stronger activation of the ROS-dependent apoptotic pathway were induced by 5FU and OXA delivered by NPs. Furthermore, it was observed that NPs were hemocompatible. Co-loaded NPs exhibited significantly higher antitumor efficiency compared to free drugs combination, indicating this co-delivery system provides great potential in cancer therapy. The results of present study also confirmed that PHBV/PLGA NPs can be served as a promising platform for the co-delivery of antitumor drugs and present a new view for treatment of cancer with reducing side effect of drugs.

A novel 5-Fluorouracil targeted delivery to colon cancer using folic acid conjugated liposomes.

The aim of this study was to develop and characterize 5-Fluorouracil (5FU) containing targeted liposomes in order to enhance the efficacy and safety of the drug. Folic acid (FA) was used as a targeting ligand. The in vitro cytotoxicity of formulation against HT-29, Caco-2, CT26, HeLa and MCF-7 cell lines was evaluated using MTT assay. Mechanism of cell death induced by targeted liposomes was further investigated via the production of reactive oxygen species (ROS), change in mitochondrial membrane potential (ΔΨm), release of cytochrome c and activity of caspase 3/7. The in vivo tumor inhibition study was also performed after administration of drug and targeted 5FU liposome. The encapsulation efficiency (EE%) of the optimized formulation was 39.71%. Particle size of liposomes was around 174 nm and the nanoparticles were found to be spherical in shape. Differential Scanning Calorimetry (DSC) results indicated that the drug remained in an amorphous state in liposomes. According to the MTT results, targeted liposomes exhibited higher cytotoxicity than 5FU and liposomal 5FU. Targeted liposomes were found to trigger necrosis in HT-29 cells; while, in HeLa cells, targeted liposomes activated apoptotic pathway by collapse of ΔΨm, increased activity of cytochrome c as well as caspases activity. in vivo results showed that targeted liposomes reduced tumor volume significantly in comparison with 5FU (169.00 mm3 tumor volume vs 326.40 mm3). From these findings, it can be concluded that folic acid targeted liposomes may provide a new platform for selective delivery of drugs to cancer cells.