Application of microphysiological systems for nonclinical evaluation of cell therapies.

Microphysiological systems (MPS) are gaining broader application in the pharmaceutical industry but have primarily been leveraged in early discovery toxicology and pharmacology studies with small molecules. The adoption of MPS offers a promising avenue to reduce animal use, improve in-vitro-to-in-vivo translation of pharmacokinetics/pharmacodynamics and toxicity correlation, and provide mechanistic understanding of model species suitability. While MPS have demonstrated utility in these areas with small molecules and biologics, cell therapeutic MPS models in drug development have not been fully explored, let alone validated. Distinguishing features of MPS, including long-term viability and physiologically relevant expression of functional enzymes, receptors, and pharmacological targets make them attractive tools for nonclinical characterization. However, there is currently limited published evidence of MPS being utilized to study the disposition, metabolism, pharmacology, and toxicity profiles of cell therapies. This review provides an industry perspective on the nonclinical application of MPS on cell therapies, first with a focus on oncology applications followed by examples in regenerative medicine.

Microphysiological systems (MPS) are advanced cell models, applied in the pharmaceutical industry to characterize novel therapies. While their application in studies of small molecule therapies has been very successful, the use of these models to study cell therapies has been limited. Cell therapies consist of cells and are living drugs, often with complex biological mechanisms of action, which can be very challenging to study. However, MPS have several features that make them attractive for studying cell therapies, including possibilities for longer-term studies and the ability to mimic physiologically relevant biological functions. MPS can mimic complex biological systems and processes, as such, the adoption of MPS offers a promising avenue to reduce the use of animals in the characterization of novel therapies. This review provides an industry perspective on current challenges and highlights opportunities for using MPS in the development of cell therapies.

Modulating tumoral exosomes and fibroblast phenotype using nanoliposomes augments cancer immunotherapy.

Cancer cells program fibroblasts into cancer associated fibroblasts (CAFs) in a two-step manner. First, cancer cells secrete exosomes to program quiescent fibroblasts into activated CAFs. Second, cancer cells maintain the CAF phenotype via activation of signal transduction pathways. We rationalized that inhibiting this two-step process can normalize CAFs into quiescent fibroblasts and augment the efficacy of immunotherapy. We show that cancer cell-targeted nanoliposomes that inhibit sequential steps of exosome biogenesis and release from lung cancer cells block the differentiation of lung fibroblasts into CAFs. In parallel, we demonstrate that CAF-targeted nanoliposomes that block two distinct nodes in fibroblast growth factor receptor (FGFR)-Wnt/ß-catenin signaling pathway can reverse activate CAFs into quiescent fibroblasts. Co-administration of both nanoliposomes significantly improves the infiltration of cytotoxic T cells and enhances the antitumor efficacy of αPD-L1 in immunocompetent lung cancer-bearing mice. Simultaneously blocking the tumoral exosome-mediated activation of fibroblasts and FGFR-Wnt/ß-catenin signaling constitutes a promising approach to augment immunotherapy.

Inhalable Lactoferrin/Chondroitin-Functionalized Monoolein Nanocomposites for Localized Lung Cancer Targeting.

Localized drug delivery to lung cancer can overcome the limitations of systemic nanocarriers including low drug amounts reaching lung tissues and severe off-target toxicity. The current work presented novel inhalable nanocomposites as noninvasive platforms for lung cancer therapy. Nanoparticulate liquid crystals (LCNPs) based on monoolein were developed for synergistic co-encapsulation of the cytotoxic chemotherapeutic drug, pemetrexed, and the phytoherbal drug, resveratrol (PEM-RES-LCNPs). For active tumor targeting, lactoferrin (LF) and chondroitin sulfate (CS), natural polymers with intrinsic tumor-targeting capabilities, were exploited to functionalize the surface of LCNPs using a layer-by-layer (LbL) self-assembly approach. To maximize their deep lung deposition, LF/CS-coated PEM-RES-LCNPs were then microencapsulated within various carriers to obtain inhalable nanocomposites via spray-drying techniques. The inhalable dry powder nanocomposites prepared using a mannitol-inulin-leucine (1:1:1 wt) mixture displayed superior in vitro aerosolization performance (2.72 µm of MMAD and 61.6% FPF), which ensured deep lung deposition. In lung cancer-bearing mice using urethane as a chemical carcinogen, the inhalable LF/CS-coated PEM-RES-LCNP nanocomposites showed superior antitumor activity as revealed by a considerable decrease of the average lung weight, reduced number and diameter of cancerous lung foci, decreased expression of VEGF-1, and increased expression of active caspase-3 as well as reduced Ki-67 expression compared to the spray-dried free PEM/RES powder mixture and positive control. Moreover, the in vivo fluorescence imaging confirmed successful lung deposition of the inhalable nanocomposites. Conclusively, the inhalable liquid crystalline nanocomposites elaborated in the current work could open new avenues for noninvasive lung cancer treatment.

Liquid crystalline assembly for potential combinatorial chemo-herbal drug delivery to lung cancer cells.

BACKGROUND: Lung cancer is the most common cancer and the leading cause of total deaths worldwide. Its classified into two major types including non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) based on the origin of abnormal lung cells as well as the smoking status of the patient. NSCLC is the most common and aggressive type of lung cancer representing 80%-85% of all cases. PURPOSE: The aim of the study was to present lyotropic liquid crystalline nanoparticles (LCNPs) as promising carriers for co-delivery of the chemotherapeutic agent, pemetrexed (PMX) and the herbal drug, resveratrol (RSV) for effective lung cancer management. METHODS: The proposed PMX-RSV-LCNPs were prepared by hydrotrope method. Hydrophobic ion pairing with cetyl trimethyl ammonium bromide (CTAB) was implemented to increase the encapsulation efficiency of the hydrophilic PMX up to 95%±3.01%. RESULTS: The tailored PMX-RSV-LCNPs exhibited a particle size of 173±0.26 nm and biphasic release pattern with a relatively initial burst release within first 3-4 hour followed by sustained release up to 24 hours. Moreover, PMX-RSV-LCNPs manifested superior concentration and time dependent cytotoxicity profile against A549 lung cancer cells with IC50 4.0628 µg/mL. Besides, the enhanced cellular uptake profile based on bioadhesive properties of glyceryl monoolein (GMO) as well as energy independent (cholesterol dependent) pattern. In-vivo evaluations against urethane induced lung cancer bearing mice demonstrated the potentiality of PMX-RSV-LCNPs in tumor growth inhibition via inhibition of angiogenesis and induction of apoptosis. The results were supported by histopathological analysis and immunohistochemical Ki67 staining. Moreover, PMX-RSV-LCNPs displayed a promising safety profile via attenuating nephro- and hepatotoxicity. CONCLUSION: PMX-RSV-LCNPs elaborated in the current study hold a great promise for lung cancer treatment.

Novel skin penetrating berberine oleate complex capitalizing on hydrophobic ion pairing approach.

Berberine hydrochloride (Brb) is a well-known herbal drug that holds a great promise in the recent years thanks to its various pharmacological actions. Currently, Brb is extensively researched as a natural surrogate with evidenced potentiality against numerous types of skin diseases including skin cancer. However, Brb's high aqueous solubility and limited permeability hinder its clinical topical application. In the current work, to enhance Brb's dermal availability, hydrophobic ion pairing approach was implemented combining the privileges of altering the solubility characteristics of Brb and the nanometric size that is usually gained during the ion pairing precipitation process. Sodium oleate (SO) was selected as the complexing agent due to its low toxicity and skin penetrating characteristics. Ion paired berberine oleate complex (Brb-OL) was prepared by simple precipitation technique. Brb-OL complex formation was confirmed by differential scanning calorimetry (DSC), infrared spectroscopy (IR), X-ray powder diffraction (XRD) and saturation solubility studies. It was found that Brb-OL complex formed at stoichiometric binding between oleate and Brb had an average particle size of 195.9 nm and zeta potential of -53.6 mV. The proposed Brb-OL showed 251-fold increase in saturation solubility in n-octanol which confirmed the augmented lipid solubility of the complex compared with free drug. Comparative in-vitro release study showed that Brb-OL complex had much slow and sustained release profile compared to that of free Brb. Furthermore, ex-vivo permeation study using rat skin revealed the enhanced skin permeation of ion-paired Brb-OL complex compared with free Brb. In-vivo study on healthy rats confirmed that topical application of hydrogels enriched with Brb-OL had superior skin penetration and deposition than free Brb as revealed by confocal microscope. Conclusively, ion pair formation between Brb and oleate lead to the formation of more lipophilic Brb-OL complex with nanometric particle size which is expected to be a major progressive step towards the development of a topical berberine formulation.

Laminated chitosan-based composite sponges for transmucosal delivery of novel protamine-decorated tripterine phytosomes: Ex-vivo mucopenetration and in-vivo pharmacokinetic assessments.

In the current study, laminated chitosan (CS):hydroxypropyl methylcellulose (HPMC) composite sponges were exploited as solid matrices for buccal delivery of tripterine phytosomes functionalized with novel mucopenetrating protamine layer (PRT-TRI-PHY). Tripterine (TRI) is a herbal drug widely investigated as a potential anticancer candidate against various types of cancers. However, clinical use of TRI is handicapped by its low oral bioavailability. To surmount TRI pharmaceutical obstacles, TRI phytosomes (TRI-PHY) were prepared using solvent evaporation technique then coated with a protamine layer via electrostatic assembly process. The developed PRT-TRI-PHY showed a nano-metric size of 250 nm and positive zeta potential (+21.6 mV). Sponges loaded with PRT-TRI-PHY demonstrated a sustained release profile with superior mucoadhesion characteristics compared with the counterparts loaded with uncoated TRI-PHY. The ex-vivo permeation study via chicken pouch mucosa revealed that sponges loaded with PRT-TRI-PHY demonstrated 2.3-folds higher flux value compared with sponges loaded with uncoated TRI-PHY. Additionally, in-vivo pharmacokinetic study in healthy rabbits revealed the significantly higher bioavailability of PRT-TRI-PHY compared with TRI-PHY with relative bioavailability of 244%. Conclusively, mucoadhesive CS-HPMC sponges loaded with a novel mucopenetrating nanocarrier, PRT-TRI-PHY, could significantly improve the absorption of tripterine via buccal mucosa which would be of prime importance for its clinical utility.

Inhalable particulate drug delivery systems for lung cancer therapy: Nanoparticles, microparticles, nanocomposites and nanoaggregates.

There is progressive evolution in the use of inhalable drug delivery systems (DDSs) for lung cancer therapy. The inhalation route offers many advantages, being non-invasive method of drug administration as well as localized delivery of anti-cancer drugs to tumor tissue. This article reviews various inhalable colloidal systems studied for tumor-targeted drug delivery including polymeric, lipid, hybrid and inorganic nanocarriers. The active targeting approaches for enhanced delivery of nanocarriers to lung cancer cells were illustrated. This article also reviews the recent advances of inhalable microparticle-based drug delivery systems for lung cancer therapy including bioresponsive, large porous, solid lipid and drug-complex microparticles. The possible strategies to improve the aerosolization behavior and maintain the critical physicochemical parameters for efficient delivery of drugs deep into lungs were also discussed. Therefore, a strong emphasis is placed on the approaches which combine the merits of both nanocarriers and microparticles including inhalable nanocomposites and nanoaggregates and on the optimization of such formulations using the proper techniques and carriers. Finally, the toxicological behavior and market potential of the inhalable anti-cancer drug delivery systems are discussed.

Self-assembled phospholipid-based phytosomal nanocarriers as promising platforms for improving oral bioavailability of the anticancer celastrol.

Celastrol (CST) is a promising natural drug of herbal origin that gained a great interest in the recent years by virtue of its wide variety of pharmacological actions. Nowadays, CST is extensively studied as a natural anticancer surrogate with a potential activity against various types of cancers. However, CST suffers from many limitations that handicapped its clinical utility such as limited aqueous solubility and poor gastrointestinal absorption which resulted into its low oral bioavailability. This work spotlights, for the first time, development of self-assembled phytosomal nanocarriers (CST-PHY) for improving CST solubility and oral bioavailability. First CST-phospholipid complex was prepared by a simple solvent evaporation technique. Formation of CST-phospholipid complex was confirmed by differential scanning calorimetry (DSC), infrared spectroscopy (IR), powder X-ray diffraction (XRD) and partition coefficient determination. After dispersion into deionized water, CST-phospholipid complex was self-assembled to form CST-PHY. The optimized CST-PHY demonstrated a nanometric particle size of 178.4±7.07nm and a negative zeta potential of -38.7±3.61mV. Comparative in-vitro release study showed the ability of phytosomes to significantly enhance CST release compared with crude drug and physical mixture. Pharmacokinetic studies in rabbits revealed significant improvement in CST-PHY oral bioavailability compared with crude CST evidenced by 4-fold increase in AUC0-8 and 5-fold increase in Cmax of CST-PHY compared with crude CST. Conclusively, the results confirmed the potential of phytosomal nanocarriers to improve CST oral delivery paving the way for its use for oral cancer therapy.

Zein-based Nanocarriers as Potential Natural Alternatives for Drug and Gene Delivery: Focus on Cancer Therapy.

Protein nanocarriers possess unique merits including minimal cytotoxicity, numerous renewable sources, and high drug-binding capability. In opposition to delivery carriers utilizing hydrophilic animal proteins, hydrophobic plant proteins (e.g, zein) have great tendency in fabricating controlled-release particulate carriers without additional chemical modification to stiffen them, which in turn evades the use of toxic chemical crosslinkers. Moreover, zein is related to a class of alcohol-soluble prolamins and generally recognized as safe (GRAS) carrier for drug delivery. Various techniques have been adopted to fabricate zein-based nanoparticulate systems including phase separation coacervation, spray-drying, supercritical anti-solvent approach, electrospinning and self-assembly. This manuscript reviews the recent advances in the zein-based colloidal nano-carrier systems such as nanospheres, nanocapsules, micelles and nanofibers with a special focus on their physicochemical characteristics and drug delivery applications.

Hyaluronate-Lipid Nanohybrids: Fruitful Harmony in Cancer Targeting.

Significant research efforts have been concerned over the past few years to design carrier systems that could specifically deliver active agents to the tumor sites, with the purposes of maximizing the therapeutic benefits and minimizing the toxic side-effects. Hyaluronic acid is a type of polysaccharide that has been extensively studied as a selective targeting ligand to cancerous cells that overexpress its specific receptor CD44. The aim of this review is to highlight the role of HA in cancer, focusing on the recent advances of HA-functionalized lipid nanoparticles towards cancer therapy and imaging.

Self-Assembled Nanocarriers Based on Amphiphilic Natural Polymers for Anti- Cancer Drug Delivery Applications.

BACKGROUND: Micellization provides numerous merits for the delivery of water insoluble anti-cancer therapeutic agents including a nanosized 'core-shell' drug delivery system. Recently, hydrophobically-modified polysaccharides and proteins are attracting much attention as micelle forming polymers to entrap poorly soluble anti-cancer drugs. METHOD: By virtue of their small size, the self-assembled micelles can passively target tumor tissues via enhanced permeation and retention effect (EPR). Moreover, the amphiphilic micelles can be exploited for active-targeted drug delivery by attaching specific targeting ligands to the outer micellar hydrophilic surface. RESULTS: Here, we review the conjugation techniques, drug loading methods, physicochemical characteristics of the most important amphiphilic polysaccharides and proteins used as anti-cancer drug delivery systems. Attention focuses on the mechanisms of tumor-targeting and enhanced anti-tumor efficacy of the encapsulated drugs. This review will highlight the remarkable advances of hydrophobized polysaccharide and protein micelles and their potential applications as anti-cancer drug delivery nanosystems. CONCLUSION: Micellar nanocarriers fabricated from amphiphilic natural polymers hold great promise as vehicles for anti-cancer drugs.

Layer-by-layer-coated lyotropic liquid crystalline nanoparticles for active tumor targeting of rapamycin.

AIM: This work spotlights on fabrication of CD44-tropic, layer-by-layer (LbL) coated, liquid crystalline nanoparticles of rapamycin (Rap-LbL-LCNPs) to enhance its water solubility and enable its anticancer use. METHODS: Rap-LCNPs were fabricated using hydrotrope method and then coated using LbL self-assembly technique. RESULTS: LbL coating strategy successfully reduced monoolein-induced hemolysis and increased LCNPs serum stability. Lyophilized Rap-LbL-LCNPs were successfully sterilized using γ-radiations. In CD44-overexpressed MDA-MB-231 cells, Rap-LbL-LCNPs demonstrated superior cytotoxicity compared with the nontargeted formulation. Rap-LbL-LCNPs showed 3.35-fold increase in bioavailability compared with free drug. Rap-LbL-LCNPs significantly inhibited tumor growth, enhanced animal survival and reduced nephrotoxic and hyperglycemic effects of Rap. CONCLUSION: LbL coating strategy of Rap-LCNPs could serve as a promising approach that facilitates Rap use in cancer therapy.

Hybrid protein-inorganic nanoparticles: From tumor-targeted drug delivery to cancer imaging.

Recently, a great interest has been paid to the development of hybrid protein-inorganic nanoparticles (NPs) for drug delivery and cancer diagnostics in order to combine the merits of both inorganic and protein nanocarriers. This review primarily discusses the most outstanding advances in the applications of the hybrids of naturally-occurring proteins with iron oxide, gadolinium, gold, silica, calcium phosphate NPs, carbon nanotubes, and quantum dots in drug delivery and cancer imaging. Various strategies that have been utilized for the preparation of protein-functionalized inorganic NPs and the mechanisms involved in the drug loading process are discussed. How can the protein functionalization overcome the limitations of colloidal stability, poor dispersibility and toxicity associated with inorganic NPs is also investigated. Moreover, issues relating to the influence of protein hybridization on the cellular uptake, tumor targeting efficiency, systemic circulation, mucosal penetration and skin permeation of inorganic NPs are highlighted. A special emphasis is devoted to the novel approaches utilizing the protein-inorganic nanohybrids in combined cancer therapy, tumor imaging, and theranostic applications as well as stimuli-responsive drug release from the nanohybrids.

Stealth, biocompatible monoolein-based lyotropic liquid crystalline nanoparticles for enhanced aloe-emodin delivery to breast cancer cells: in vitro and in vivo studies.

Recently, research has progressively highlighted on clues from conventional use of herbal medicines to introduce new anticancer drugs. Aloe-emodin (AE) is a herbal drug with promising anticancer activity. Nevertheless, its clinical utility is handicapped by its low solubility. For the first time, this study aims to the fabrication of surface-functionalized polyethylene glycol liquid crystalline nanoparticles (PEG-LCNPs) of AE to enhance its water solubility and enable its anticancer use. Developed AE-PEG-LCNPs were optimized via particle size and zeta potential measurements. Phase behavior, solid state characteristics, hemocompatibility, and serum stability of LCNPs were assessed. Sterile formulations were developed using various sterilization technologies. Furthermore, the potential of the formulations was investigated using cell culture, pharmacokinetics, biodistribution, and toxicity studies. AE-PEG-LCNPs showed particle size of 190 nm and zeta potential of -49.9, and PEGylation approach reduced the monoolein hemolytic tendency to 3% and increased the serum stability of the nanoparticles. Sterilization of liquid and lyophilized AE-PEG-LCNPs via autoclaving and γ-radiations, respectively, insignificantly affected the physicochemical properties of the nanoparticles. Half maximal inhibitory concentration of AE-PEG-LCNPs was 3.6-fold lower than free AE after 48 hours and their cellular uptake was threefold higher than free AE after 24-hour incubation. AE-PEG-LCNPs presented 5.4-fold increase in t1/2 compared with free AE. Biodistribution and toxicity studies showed reduced AE-PEG-LCNP uptake by reticuloendothelial system organs and good safety profile. PEGylated LCNPs could serve as a promising nanocarrier for efficient delivery of AE to cancerous cells.

Lyophilized phytosomal nanocarriers as platforms for enhanced diosmin delivery: optimization and ex vivo permeation.

Diosmin (DSN) is an outstanding phlebotonic flavonoid with a tolerable potential for the treatment of colon and hepatocellular carcinoma. Being highly insoluble, DSN bioavailability suffers from high inter-subject variation due to variable degrees of permeation. This work endeavored to develop novel DSN loaded phytosomes in order to improve drug dissolution and intestinal permeability. Three preparation methods (solvent evaporation, salting out, and lyophilization) were compared. Nanocarrier optimization encompassed different soybean phospholipid (SPC) types, different solvents, and different DSN:SPC molar ratios (1:1, 1:2, and 1:4). In vitro appraisal encompassed differential scanning calorimetry, infrared spectroscopy, particle size, zeta potential, polydispersity index, transmission electron microscopy, drug content, and in vitro stability. Comparative dissolution studies were performed under sink versus non-sink conditions. Ex vivo intestinal permeation studies were performed on rats utilizing noneverted sac technique and high-performance liquid chromatography analysis. The results revealed lyophilization as the optimum preparation technique using SPC and solvent mixture (Dimethyl sulphoxide:t-butylalchol) in a 1:2 ratio. Complex formation was contended by differential scanning calorimetry and infrared data. Optimal lyophilized phytosomal nanocarriers (LPNs) exhibited the lowest particle size (316 nm), adequate zeta-potential (-27 mV), and good in vitro stability. Well formed, discrete vesicles were revealed by transmission electron microscopy, drug content, and in vitro stability. Comparative dissolution studies were performed. LPNs demonstrated significant enhancement in DSN dissolution compared to crude drug, physical mixture, and generic and brand DSN products. Permeation studies revealed 80% DSN permeated from LPNs via oxygenated rat intestine compared to non-detectable amounts from suspension. In this study, LPNs (99% drug loading) could be successfully tailored for DSN with improved dissolution and permeation characteristics, which is promising for lowering the influence of exogenous factors and increasing drug delivery.