PEGylated Liposomes Loaded with Carbamate Inhibitor ANP0903 Trigger Apoptosis by Enhancing ER Stress in HepG2 Cancer Cells.

Liver cancer is one of the most common causes of cancer death worldwide. In recent years, substantial progress has been made in the development of systemic therapies, but there is still the need for new drugs and technologies that can increase the survival and quality of life of patients. The present investigation reports the development of a liposomal formulation of a carbamate molecule, reported as ANP0903, previously tested as an inhibitor of HIV-1 protease and now evaluated for its ability to induce cytotoxicity in hepatocellular carcinoma cell lines. PEGylated liposomes were prepared and characterized. Small, oligolamellar vesicles were produced, as demonstrated by light scattering results and TEM images. The physical stability of the vesicles in biological fluids was demonstrated in vitro, alongside the stability during storage. An enhanced cellular uptake was verified in HepG2 cells treated with liposomal ANP0903, resulting in a greater cytotoxicity. Several biological assays were performed to elucidate the molecular mechanisms explaining the proapoptotic effect of ANP0903. Our results allow us to hypothesize that the cytotoxic action in tumor cells is probably due to the inhibition of the proteasome, resulting in an increase in the amount of ubiquitinated proteins within the cells, which in turn triggers activation of autophagy and apoptosis processes, resulting in cell death. The proposed liposomal formulation represents a promising approach to deliver a novel antitumor agent to cancer cells and enhance its activity.

Nanodesign of new self-assembling core-shell gellan-transfersomes loading baicalin and in vivo evaluation of repair response in skin.

Gellan nanohydrogel and phospholipid vesicles were combined to incorporate baicalin in new self-assembling core-shell gellan-transfersomes obtained by an easy, scalable method. The vesicles were small in size (~107 nm) and monodispersed (P.I. ≤ 0.24), forming a viscous system (~24 mPa/s) as compared to transfersomes (~1.6 mPa/s), as confirmed by rheological studies. Gellan was anchored to the bilayer domains through cholesterol, and the polymer chains were distributed onto the outer surface of the bilayer, thus forming a core-shell structure, as suggested by SAXS analyses. The optimal carrier ability of core-shell gellan-transfersomes was established by the high deposition of baicalin in the skin (~11% in the whole skin), especially in the deeper tissue (~8% in the dermis). Moreover, their ability to improve baicalin efficacy in anti-inflammatory and skin repair tests was confirmed in vivo in mice, providing the complete skin restoration and inhibiting all the studied inflammatory markers.

Enhancing Topical Delivery of Resveratrol through a Nanosizing Approach.

Resveratrol is a naturally occurring polyphenol with strong antioxidant and free radical scavenging properties, recently proposed as a therapeutic agent for skin diseases. In this study, we investigated the possibility of improving the dermal bioavailability of the poorly water-soluble drug resveratrol by nanocrystal technology. To this purpose, nanosuspensions were prepared by the wet media milling technique, using Poloxamer 188 or Tween 80 as stabilizers, and characterized by means of both solid state and morphological and dimensional studies. All analytical data demonstrated that neither a modification of the drug crystalline pattern nor the isomerization of the trans double bond were observed after the wet media milling particle size reduction process, which produced rounded and smooth nanocrystals with a mean diameter ranging between 0.2-0.3 µm. Resveratrol skin delivery from nanosuspension formulations was evaluated by the pig ear skin model via tape stripping. Results of the experiments showed that after application of nanosuspension formulations, higher amounts of resveratrol could penetrate the skin at deeper levels compared to drug coarse suspensions. The antioxidant activity of resveratrol in nanocrystals was assessed by the DPPH assay, which demonstrated that the size reduction process as well as the formulation compositions did not modify the drug antioxidant activity.

Needle-free jet injection of intact phospholipid vesicles across the skin: a feasibility study.

Needle-free liquid jet injectors are devices developed for the delivery of pharmaceutical solutions through the skin. In this paper, we investigated for the first time the ability of these devices to deliver intact lipid vesicles. Diclofenac sodium loaded phospholipid vesicles of two types, namely liposomes and transfersomes, were prepared and fully characterized. The lipid vesicles were delivered through a skin specimen using a jet injector and the collected samples were analyzed to assess vesicle structural integrity, drug retention and release kinetics after the injection. In this regard, data concerning size, size distribution, surface charge of vesicles and bilayer integrity and thickness, before and after the injections, were measured by dynamic light scattering experiments, cryo-electron microscopy, and X-ray scattering techniques. Finally, the effect of vesicle fast jet injection through the skin on drug release kinetics was checked by in vitro experiments. The retention of the morphological, physico-chemical, and technological features after injection, proved the integrity of vesicles after skin crossing as a high-speed liquid jet. The delivery of undamaged vesicular carriers beneath the skin is of utmost importance to create a controlled release drug depot in the hypoderm, which may be beneficial for several localized therapies. Overall results reported in this paper may broaden the range of application of liquid jet injectors to lipid vesicle based formulations thus combining beneficial performance of painless devices with those of liposomal drug delivery systems.

Pegylated-liposomes increase the efficacy of Idelalisib in lymphoma B-cells.

New drugs and technologies are continuously developed to improve the efficacy and minimize the critical side effects of cancer treatments. The present investigation focuses on the development of a liposomal formulation for Idelalisib, a small-molecule kinase inhibitor approved for the treatment of lymphoid malignancies. Idelalisib is a potent and selective antitumor agent, but it is not indicated nor recommended for first-line treatment due to fatal and serious toxicities. Herein, liposomes are proposed as a delivery tool to improve the therapeutic profile of Idelalisib. Specifically, PEGylated liposomes were prepared, and their physicochemical and technological features were investigated. Light-scattering spectroscopy and cryo-transmission electron microscopy revealed nanosized unilamellar vesicles, which were proved to be stable in storage and in simulated biological fluids. The cytotoxicity of the liposome formulation was investigated in a human non-Hodgkin's lymphoma B cell line. Idelalisib was able to induce death of tumor cells if delivered by the nanocarrier system at increased efficacy. These findings suggest that combining Idelalisib and nanotechnologies may be a powerful strategy to increase the antitumor efficacy of the drug.

Boosting antigen-specific T cell activation with lipid-stabilized protein nanoaggregates.

Vaccines based on protein antigens have numerous advantages over inactivated pathogens, including easier manufacturing and improved safety. However, purified antigens are weakly immunogenic, as they lack the spatial organization and the associated 'danger signals' of the pathogen. Formulating vaccines as nanoparticles enhances the recognition by antigen presenting cells, boosting the cell-mediated immune response. This study describes a nano-precipitation method to obtain stable protein nanoaggregates with uniform size distribution without using covalent cross-linkers. Nanoaggregates were formed via microfluidic mixing of ovalbumin (OVA) and lipids in the presence of high methanol concentrations. A purification protocol was set up to separate the nanoaggregates from OVA and liposomes, obtained as byproducts of the mixing. The nanoaggregates were characterized in terms of morphology, ζ-potential and protein content, and their interaction with immune cells was assessed in vitro. Antigen-specific T cell activation was over 6-fold higher for nanoaggregates compared to OVA, due in part to the enhanced uptake by immune cells. Lastly, a two-dose immunization with nanoaggregates in mice induced a significant increase in OVA-specific CD8+ T splenocytes compared to soluble OVA. Overall, this work presents for the first time the microfluidic production of lipid-stabilized protein nanoaggregates and provides a proof-of-concept of their potential for vaccination.

Beclomethasone loaded liposomes enriched with mucin: A suitable approach for the control of skin disorders.

Inflammatory skin disorders are the fourth leading cause of chronic non-fatal conditions, which have a serious impact on the patient quality of life. Due to their treatment with conventional corticosteroids, which often result in poor therapeutic efficacy, relapses and systemic side effects from prolonged therapy, these diseases represent a global burden that negatively impacts the global economy. To avoid these problems and optimize corticosteroid benefits, beclomethasone was loaded into liposome formulations specifically tailored for skin delivery. These formulations were enhanced with mucin (0.1 and 0.5 % w/v) to further ensure prolonged formulation permanence at the site of application. The addition of 0.5 % w/v mucin resulted in the formation of small unilamellar vesicles and multicompartment vesicles. Liposomes and 1mucin-liposomes were smaller (∼48 and ∼61 nm, respectively) and more monodispersed (PI ∼ 0.14 and ∼ 0.17, respectively) than 5mucin-liposomes, which were larger (∼137 nm), slightly polydispersed (PI ∼ 0.23), and less stable during storage (4 months in the dark at 25 °C). Liposomes were negatively charged (∼ -79 mV) irrespective of their composition, and capable of incorporating high amount of beclomethasone (∼ 80 %). In vitro studies on skin fibroblasts and keratinocytes confirmed the high biocompatibility of all formulations (viability ≥ 95 %). However, the use of mucin-liposomes resulted in higher efficacy against nitric oxide production and free radical damage. Finally, topical applications using 12-O-tetradecanoylphorbol-13-acetate-injured skin in vivo experiments showed that only the mucin-enriched formulations could restore healthy conditions within 4 days, underscoring promise as a treatment for skin disorders.

Drug silica nanocomposite: preparation, characterization and skin permeation studies.

The aim of this work was to evaluate silica nanocomposites as topical drug delivery systems for the model drug, caffeine. Preparation, characterization, and skin permeation properties of caffeine-silica nanocomposites are described. Caffeine was loaded into the nanocomposites by grinding the drug with mesoporous silica in a ball mill up to 10 h and the efficiency of the process was studied by XRPD. Formulations were characterized by several methods that include FTIR, XRPD, SEM and TEM. The successful loading of caffeine was demonstrated by XRPD and FTIR. Morphology was studied by SEM that showed particle size reduction while TEM demonstrated formation of both core-shell and multilayered caffeine-silica structures. Solid-state NMR spectra excluded chemical interactions between caffeine and silica matrix, thus confirming that no solid state reactions occurred during the grinding process. Influence of drug inclusion in silica nanocomposite on the in vitro caffeine diffusion into and through the skin was investigated in comparison with a caffeine gel formulation (reference), using newborn pig skin and vertical Franz diffusion cells. Results from the in vitro skin permeation experiments showed that inclusion into the nanocomposite reduced and delayed caffeine permeation from the silica nanocomposite in comparison with the reference, independently from the amount of the tested formulation.

Nanosuspension-Based Dissolvable Microneedle Arrays to Enhance Diclofenac Skin Delivery.

Applying a formulation on the skin represents a patient-acceptable and therapeutically effective way to administer drugs locally and systemically. However, the stratum corneum stands as an impermeable barrier that only allows a very limited number of drugs to be distributed in the underlying tissues, limiting the feasibility of this administration route. Microneedle arrays are minimally invasive platforms that allow the delivery of drugs within/across the skin through the temporary mechanical disruption of the stratum corneum. In this work, microneedle arrays were combined with nanosuspensions, a technology for solubility enhancement of water insoluble molecules, for the skin delivery of diclofenac. Nanosuspensions were prepared using a top-down method and loaded in the tips of 500 µm or 800 µm high microneedles. The quality of the combined platform was assessed using electron microscopy and spectroscopic and calorimetry techniques, demonstrating the ability to load high amounts of the hydrophobic drug and the compatibility between excipients. Lastly, the application of nanosuspension-loaded microneedles on the skin in vitro allowed the delivery of diclofenac within and across the stratum corneum, proving the potential of this combination to enhance skin delivery of scarcely soluble drugs.

Needle-Free Jet Injectors and Nanosuspensions: Exploring the Potential of an Unexpected Pair.

Needle-free liquid jet injectors are medical devices used to administer pharmaceutical solutions through the skin. Jet injectors generate a high-speed stream of liquid medication that can puncture the skin and deliver the drug to the underlying tissues. In this work, we investigated the feasibility of using liquid jet injectors to administer nanosuspensions, assessing the impact of the jet injection on their pharmaceutical and physicochemical properties. For this purpose, the model drug diclofenac was used to prepare a set of nanosuspensions, stabilized by poloxamer 188, and equilibrated at different pHs. The hydrodynamic diameter and morphology of the nanocrystals were analyzed before and after the jet injection across porcine skin in vitro, together with the solubility and release kinetics of diclofenac in a simulated subcutaneous environment. The efficacy of the jet injection (i.e., the amount of drug delivered across the skin) was evaluated for the nanosuspension and for a solution, which was used as a control. Finally, the nanosuspension was administered to rats by jet injector, and the plasma profile of diclofenac was evaluated and compared to the one obtained by jet injecting a solution with an equal concentration. The nanosuspension features were maintained after the jet injection in vitro, suggesting that no structural changes occur upon high-speed impact with the skin. Accordingly, in vivo studies demonstrated the feasibility of jet injecting a nanosuspension, reaching relevant plasma concentration of the drug. Overall, needle-free jet injectors proved to be a suitable alternative to conventional syringes for the administration of nanosuspensions.

Nanocrystals as an effective strategy to improve Pomalidomide bioavailability in rodent.

Pomalidomide (POM) is an FDA-approved immunomodulatory imide drug (IMiDs) an it is effectively used in the treatment of multiple myeloma. IMiDs are analogs of the drug thalidomide and they have been repurposed for the treatment of several diseases such as psoriatic arthritis and Kaposi Sarcoma. In recent years, IMiDs have been also evaluated as a new treatment for neurological disorders with an inflammatory and neuroinflammatory component. POM draws particular interest for its potent anti-TNF-α activity at significantly lower concentrations than the parent compound thalidomide. However, POM's low water solubility underpins its low gastrointestinal permeability resulting in irregular and poor absorption. The purpose of this work was to prepare a POM nanocrystal-based formulation that could efficiently improve POM's plasma and brain concentration after intraperitoneal injection. POM nanocrystals prepared as a nanosuspension by the media milling method showed a mean diameter of 219 nm and a polydispersity index of 0.21. POM's nanocrystal solubility value (22.97 µg/mL) in phosphate buffer was about 1.58 folds higher than the POM raw powder. Finally, in vivo studies conducted in adult Male Sprague-Dawley rats indicated that POM nanocrystal ensured higher and longer-lasting drug levels in plasma and brain when compared with POM coarse suspension.

Delivery of beclomethasone dipropionate nanosuspensions with an electronic cigarette.

The aim of this work was to ascertain the ability of electronic nicotine delivery systems (ENDS) to deliver drug nanocrystals through the produced aerosol. A nanocrystal nanosuspension of beclomethasone dipropionate, a synthetic chlorinated corticosteroid diester commonly used by inhalation in the treatment of asthma and chronic obstructive pulmonary disease, was prepared with a wet media milling technique using Poloxamer 188 as stabilizer. The obtained nanosuspension was thoroughly characterized by different techniques: transmission electron microscopy, photon correlation spectroscopy, X-ray powder diffractometry and Fourier transform infrared spectroscopy. The nanosuspension was then loaded in the cartomizer of the electronic cigarette and the produced aerosol was collected and analysed, confirming the presence of drug nanocrystals. The results of this study suggested the possible alternative use of ENDS as medical device for the delivery of poorly soluble drugs.

Pulmonary Delivery of Curcumin and Beclomethasone Dipropionate in a Multicomponent Nanosuspension for the Treatment of Bronchial Asthma.

Curcumin has shown a potential extraordinary activity as an add-on ingredient in asthma treatment, due to its immunomodulatory and anti-inflammatory mechanism of action. However, its low water solubility and bioavailability lead to a poor therapeutic effect, which can be overcome by its formulation as nanocrystals. The aim of this study was to prepare a multicomponent formulation for the delivery of curcumin (CUR) and beclomethasone dipropionate (BDP) into the lungs as water-based nanosuspensions (NS). Single component formulations (CUR-NS, BDP-NS) and a multicomponent formulation (CUR+BDP-NS) were prepared through a wet ball media milling technique, using P188 as a non-toxic stabilizer. Characterization was carried out in terms of size, size distribution, zeta potential, nanocrystals morphology, and solid-state properties. Moreover, the inhalation delivery efficiency was studied with Next Generation Impactor (NGI, Apparatus E Ph. Eu). CUR-NS was optimized and showed a long-term stability and improved nanocrystals apparent solubility. The three formulations exhibited a nanocrystal mean diameter in the range of 200-240 nm and a homogenous particle size distribution. Aggregation or sedimentation phenomena were not observed in the multicomponent formulation on 90 days storage at room temperature. Finally, the nebulization tests of the three samples showed optimal aerodynamic parameters and MMAD < 5 µm.

Loading of Beclomethasone in Liposomes and Hyalurosomes Improved with Mucin as Effective Approach to Counteract the Oxidative Stress Generated by Cigarette Smoke Extract.

In this work beclomethasone dipropionate was loaded into liposomes and hyalurosomes modified with mucin to improve the ability of the payload to counteract the oxidative stress and involved damages caused by cigarette smoke in the airway. The vesicles were prepared by dispersing all components in the appropriate vehicle and sonicating them, thus avoiding the use of organic solvents. Unilamellar and bilamellar vesicles small in size (~117 nm), homogeneously dispersed (polydispersity index lower than 0.22) and negatively charged (~-11 mV), were obtained. Moreover, these vesicle dispersions were stable for five months at room temperature (~25 °C). In vitro studies performed using the Next Generation Impactor confirmed the suitability of the formulations to be nebulized as they were capable of reaching the last stages of the impactor that mimic the deeper airways, thus improving the deposition of beclomethasone in the target site. Further, biocompatibility studies performed by using 16HBE bronchial epithelial cells confirmed the high biocompatibility and safety of all the vesicles. Among the tested formulations, only mucin-hyalurosomes were capable of effectively counteracting the production of reactive oxygen species (ROS) induced by cigarette smoke extract, suggesting that this formulation may represent a promising tool to reduce the damaging effects of cigarette smoke in the lung tissues, thus reducing the pathogenesis of cigarette smoke-associated diseases such as chronic obstructive pulmonary disease, emphysema, and cancer.

In Vitro Skin Permeation of Idebenone from Lipid Nanoparticles Containing Chemical Penetration Enhancers.

Lipid nanoparticles (LNPs) have been proposed as carriers for drug skin delivery and targeting. As LNPs effectiveness could be increased by the addition of chemical penetration enhancers (PE), in this work, the feasibility of incorporating PE into LNPs to improve idebenone (IDE) targeting to the skin was investigated. LNPs loading IDE 0.7% w/w were prepared using hydrophilic (propylene glycol, PG, 10% w/w or N-methylpyrrolidone, NMP, 10% w/w) and/or lipophilic PE (oleic acid, OA, 1% w/w; isopropyl myristate, IPM, 3.5% w/w; a mixture of 0.5% w/w OA and 2.5% w/w IPM). All LNPs showed small sizes (<60 nm), low polydispersity index and good stability. According to the obtained results, IDE release from LNPs was not the rate-limiting step in IDE skin penetration. No IDE permeation was observed through excised pigskin from all LNPs, while the greatest increase of IDE penetration into the different skin layers was obtained using the mixture OA/IPM. The antioxidant activity of IDE-loaded LNPs, determined by the oxygen radical absorbance capacity assay, was greater than that of free IDE. These results suggest that the use of suitable PE as LNPs components could be regarded as a promising strategy to improve drug targeting to the skin.

Efficacy of a resveratrol nanoformulation based on a commercially available liposomal platform.

Scalability is one of the important factors slowing down or even impeding the clinical translation of nanoparticle-based systems. The latter need to be manufactured at a high level of quality, with batch-to-batch reproducibility, and need to be stable after the manufacturing process, during long-term storage and upon clinical administration. In this study, a vesicular formulation intended for cutaneous applications was developed by the easy reconstitution of a commercially available liposomal platform. Resveratrol, a naturally occurring compound with potent antioxidant activity, and Tween80, a hydrophilic non-ionic surfactant, were included in the formulation. The physico-chemical properties of the vesicles were assessed using light scattering and cryogenic transmission electron microscopy. Nanosized (around 80 nm) spherical and elongated, unilamellar vesicles were produced, with remarkable storage stability. The incorporation of resveratrol in the vesicular system did not alter its strong antioxidant activity, as demonstrated by antioxidant colorimetric assays (DPPH and FRAP). Furthermore, the resveratrol liposomes were cytocompatible with fibroblasts and capable of protecting skin cells from oxidative stress by reducing both endogenous and chemically induced reactive oxygen species more effectively than free resveratrol. Therefore, the proposed formulation, based on the use of a commercially available liposomal platform, represents an easy-to-prepare, reproducible, up-scaled and efficient means of delivering resveratrol and potentiating its biological activity in vitro.

Nanoliposomes@Transcutol for In Vitro Skin Delivery of 8-Methoxypsoralen.

8-methoxypsoralen is the most common drug in psoralen plus ultraviolet light irradiation therapy for the treatment of severe psoriasis. Despite of the efficacy, its classic oral administration leads to several serious adverse effects. However, the topical psoralen application produces a drug skin accumulation lower than that obtained by oral administration, due to the drug low skin permeability. In this paper, 8-methoxypsoralen loaded Penetration Enhancer-containing Vesicles were prepared using soy phosphatidylcholine and the penetration enhancer Transcutol® (5% or 10%) and characterized in terms of size, polydispersity index, zeta potential and encapsulation efficiency. No statistically significant differences in both size (~135 nm) and encapsulation efficiency (~65%) were found for different Transcutol® concentration. Transdermal delivery study assessed by Franz diffusion cells, showed that the 8-methoxypsoralen mainly accumulated into the stratum corneum. Moreover, after Penetration Enhancer-containing Vesicles application, the drug recovered in this layer is almost double of that delivered by conventional liposomes, while no significant difference was found from the different Transcutol® concentrations. Finally, biocompatibility checked by an MTT assay, demonstrated that the incubation of human keratinocytes for 24 h with 8-methoxypsoralen loaded Penetration Enhancer-containing Vesicles did not significantly reduce cell viability.

Nanocrystals as Tool to Enhance Stigmasterol Oral Bioavailability.

Phytosterols are sterols naturally occurring in plant cells and well known for their cholesterollowering activity, as witnessed by the large number of food supplements based on these functional ingredients available on the market. However, the marked hydrophobic character of phytosterols makes their solubility in biological fluids extremely low, with disadvantageous consequences on the bioavailability and therapeutic efficacy. In this work, we explore the effect of particle size reduction on the water solubility of stigmasterol, one of the most abundant phytosterols, through the formulation of nanocystals. A robust, top-down production process was employed to prepare stigmasterol nanocrystals, subsequently characterized by thermal and spectroscopic techniques. When formulated as nanocrystals, the solubility of stigmasterol in water and in simulated gastro-intestinal fluids was boosted compared to the raw material. The increased solubility of stigmasterol nanocrystals makes such formulation a promising candidate for the development of medicinal/nutraceutical products with enhanced bioavailability.

Resveratrol and artemisinin eudragit-coated liposomes: A strategy to tackle intestinal tumors.

Resveratrol and artemisinin, two naturally occurring compounds with a wide range of biological activities, have been reported to exert antitumor effects against several types of cancer. In this work, Eudragit-coated liposomes were developed to safely transport resveratrol and artemisinin through the gastrointestinal tract and target the intestine. The physico-chemical properties of the Eudragit-coated liposomes were assessed by light scattering and cryogenic transmission electron microscopy. Nanosized (around 100 nm), spherical or elongated, unilamellar vesicles were produced. The protective effect of the Eudragit coating was confirmed by assessing the physical stability of the vesicles in fluids mimicking the gastrointestinal environment. Furthermore, the vesicles were found to exert a pro-oxidant activity in intestinal adenocarcinoma cells, which resulted in a marked mortality due to the generation of reactive oxygen species (ROS). A time- and dose-dependent cell growth inhibitory effect was detected, with elevated ROS levels when resveratrol and artemisinin were combined. Therefore, the proposed formulations may represent a valuable means to counteract intestinal tumor growth.

Proniosomal Formulation Encapsulating Pomegranate Peel Extract for Nutraceutical Applications.

In this study, pomegranate peel as a traditional natural remedy was extracted and encapsulated in proniosomal systems in order to improve its stability against harsh environmental conditions. Pomegranate peel was extracted by using sonication as a green extraction technology and the antioxidant activity of the obtained extract was evaluated to be 85.37% by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Proniosomal powder was prepared based on the slurry method with a mixture of non-ionic surfactants namely span 60 and tween 20 in combination with cholesterol as a bilayer stabilizer. Proniosome-derived niosomes were achieved by hydration of the powder with water. The obtained vesicles were evaluated for their particle size, morphological observations, entrapment efficiency, cytotoxicity assay, DPPH antioxidant activity and, physical stability at 4 °C for 28 days. The results demonstrated that the proniosome-derived niosomes were of small size (198.16 nm for unloaded and 411.3 for extract loaded), quite homogeneous (PDI = 0.188 for unloaded and 0.216 for loaded) with highly negative charged spherical vesicles and showed appropriate physical stability during the time of storage. The encapsulation efficiency was 68.43±0.24% and the cytotoxicity assay proved that the formulations were not toxic against 3T3 fibroblast cells in the applied concentration.