Area I: Percutaneous Delivery of small molecules and peptides

Percutaneous delivery provides advantages for skin diseases where the target site is present in the deep epidermis (e.g. fungal, bacterial, viral- infections, psoriasis, dermatitis and skin cancers like melanoma). In addition, percutaneous delivery offers several advantages over the conventional oral and intravenous dosage forms such as prevention of the first-pass metabolism, minimization of pain and possible controlled release of drugs. However, a foremost layer of the skin, stratum corneum (SC), acts as the main barrier between the body and the environment and limits the delivery of most drugs. To overcome SC barrier, different nanoparticles such as SLN, NLC, and polymeric nanoparticles were investigated for percutaneous delivery of various small molecules and peptides.

A) Bilayered Polymeric Nanoparticles for Combination Therapy:

Bilayered polymeric nanoparticles (NPS) are prepared for combination therapy. These NPS are prepared using poly(lactic-co-glycolic acid (PLGA) and chitosan where one drug is encapsulated in PLGA core while another drug is encapsulated into chitosan coat. However, these nanoparticles were unable to reach to the deeper skin layers. Therefore the surface of NPS was modified with oleic acid (OA), FDA approved penetration enhancer. During our human skin permeation experiments, we have noted that the surface modification of the NPS with oleic acid (NPS-OA) is responsible for the significant improvement in the spantide (SP), and ketoprofen (KP) delivery to the deeper skin layers.

SP is an anti-inflammatory peptide (MW 1668.76) that antagonizes the neurokinin-1 receptors and thus inhibits inflammatory response associated with substance-P. KP is a potent non-steroidal anti-inflammatory drug (NSAID) which inhibits arachidonic acid metabolism by potent inhibitory action on cyclooxygenase.

B) Nanostructured Lipid Carriers for Percutaneous Drug Delivery:

Various lipid carriers such as liposomes, solid lipid nanoparticles (SLN) have been well studied for percutaneous drug delivery. However, liposomes have limitations in their a) physical stability problems with liposomes and b) high costs for effective large-scale production. Similarly, SLNs have the limitation of drug expulsion from the carrier and limited drug loading capacity. To overcome this, the second generation of lipid nanoparticles, nanostructured lipid carriers (NLCs) have been developed using the blend of both solid lipids and liquid lipids (oils). NLCs has higher loading capacity and lower drug leaching in storage compared to SLN.

In our efforts to enhance the delivery of NLCs into the deep epidermis, we have already shown that a well-known Cell penetrating peptide (CPPs), transactivating transcriptional activator (TAT), when linked to nanoparticles, has the potential to carry the payloads across the skin layers. We are screening different CPPs for enhancing the drug delivery to the deep epidermis. Further, this approach was used to deliver a peptide, spantide II, and small molecule, ketoprofen together for the treatment of skin diseases like allergic contact dermatitis (ACD).

Area II : Development of Animal Models for Inflammatory Skin Diseases

A) Allergic Contact Dermatitis (ACD):

Allergic contact dermatitis (ACD) is one of the most prevalent occupational skin diseases. ACD is may result from the interaction of xenobiotics with the immune system. ACD is regarded as an emergence of immunotoxicity in humans. Many chemicals have been shown to cause skin sensitization. In common with other forms of allergy, ACD develops in two phases which are defined operationally as induction and elicitation. Induction of skin sensitization is initiated by topical exposure of the chemical allergen to an individual sufficient to induce a cutaneous immune response of the necessary vigor, known as sensitization. If the sensitized individual is now exposed subsequently, at the same or a different skin site, to the inducing chemical allergen then a more vigorous secondary immune response will be provoked at the point of contact. This, in turn, initiates the cutaneous inflammatory reaction known as ACD. This model was sensitized and challenged using 2,4-dinitrofluorobenzene (DNFB).

B) Psoriasis-like model:

Psoriasis is a chronic inflammatory skin disease and is characterized by erythematous plaques, excessive growth and aberrant differentiation of keratinocytes along with increases in angiogenesis and inflammatory cell infiltrate. Most commonly used model for investigation of psoriasis is a xenograft model, in which immunodeficient mice are transplanted with human psoriasis-prone skin. However, these kinds of experiments are laborious, expensive and require considerable expertise and technical skills.Therefore a psoriasis plaque like the model was developed using topical application of imiquimod (IMQ) on mice back skin. This model showed the most features of human psoriasis where cytokines like IL-23 and IL-17 are involved.

Area III: Delivery of Anticancer Drugs

This area involves the study of molecular mechanisms involved in anti-cancer therapy using mouse xenograft tumor models. Various lung xenograft models (orthotopic, i.v. administration of cells, s.c. model) are available in the laboratory. The study involves the use of novel PPAR-gamma agonists against non-small cell lung cancer and understanding their mechanism of action either alone or in combination with Taxotere. Novel approaches for the delivery of these anticancer drugs and are used with a brief outline:

Aerosols:

Formulation of HFA based (134-a and 227) metered dose inhalers with anticancer drugs, proteins and Cox-2 inhibitors.

Nebulization of Cox-2 inhibitors to lungs using lung tumor models. A nose only chamber is available and has been extensively used to evaluate for deposition and efficacy of aerosols in mice.

Formulations of nasal formulations using various polymers and their assessment for droplet size, viscosity and surface tension using the statistical design of experiments.

Liposomal/Nanoparticle drug delivery And Overcoming Stromal Barriers:

Development of various anticancer drugs-antibody conjugates, a liposome containing drugs, liposome-antibody conjugates and nanoparticle formulations for cancer drug delivery in combination with immunotoxins.

One important part of our research is to enhance delivery of liposomes to tumors cells by overcoming stromal barriers by using drugs like Telmisartan and Losartan and withaferin. Apart from this, our research group is also delivering siRNA to tumor cells by using liposomal delivery systems. Imaging of targeted liposomes is also done with using various chemoluminescence and bioluminescence markers.

Area IV: Alternative to use of animals using 3D In vitro culture models

Another component of research in Dr. Sachdeva’s laboratory is the use of various 3D in vitro cultures as an alternative to animals. In this area, two projects are being pursued. The role of 3D cultures as models to study skin irritation has already been demonstrated and various publications are already available in this area.

A) In vitro 3D culture model for wound healing:

Presently there is no wound healing model available in vitro and most studies are being conducted in vivo. In our laboratory, we are developing a 3D human wound healing model using the EFT-300 cultures from Mattek Corp. Currently, the model is being developed using either various corrosive chemicals (e.g. Sulphuric acid, acrylic acid, potassium hydroxide) to induce a wound or heat to induce a burn wound. Various histological changes along with the role of various growth factors and cytokines are also being monitored to validate such a model. EFT-300 model consists of epidermis and dermis which are very important to understand the natural mechanism of wound healing to establish normal equilibrium of the skin.

B) In vitro tumor 3D culture model:

This project involves the development of in-vitro tumor model which is more predictive of disease states and drug responses. Algimatrix obtained from Invitrogen is currently being used as the matrix to grow tumor cells as spheroids which will then be treated with various drugs/formulations and to perform various mechanistic studies. The objective is to show a correlation between in vitro and in vivo studies and currently studies are in progress with lung tumor cells. Algimatrix is pure, non-toxic, has a better nutrient delivery without damage to cells.

Area V: Synthesis and characterization of various PEG-cholecalciferol conjugates for ocular drug delivery

Dry eye Disease (DED) is an ocular disease caused by hyperosmolarity of tears which is manifested by discomfort and visual disturbances consequently damaging the ocular surface. Topical administration is the most favored route for the treatment of DED, however, frequent drainage from the ocular surface upon administration and poor corneal permeability requires frequent administration of formulations. So there is a need for innovative formulation strategies that can overcome these shortcomings. We are designing of cholecalciferol PEG conjugates which can function as an ester prodrug for cholecalciferol as well as self-assemble into nanomicelles due to hydrophilic PEG2000 for the ocular delivery of various drugs such as tacrolimus to treat DED. A mouse model of DED was developed using benzalkonium chloride treatment and therapeutic efficacy of cholecalciferol conjugates was evaluated. Progression of DED was monitored by assessing tear pH and corneal staining scores. We observed significant improvement in DED in terms of corneal grading score (1 vs 4) and reduction of inflammatory cells

CORNEAL PENETRATION STUDY:

Fresh porcine eyes were obtained from Bradley Country Store in Tallahassee, FL (slaughterhouse). The anterior portion of the eye was cannulated using a 27 g needle and first flushed and inflated with keratinocyte serum-free medium (KSFM with pituitary extract and EGF) followed by the medium flow rate of 1-2 microlitre/min for the period of study at 37°C. Different formulations were prepared with Coumarin-6 as a tracer dye and applied to eyeballs. Confocal laser scanning microscopy with z-stacking (10μ intervals) was performed. Fluorescence intensities at different depths of cornea showed significant differences in the different formulations. Nanomicelle gel showed deeper penetration and also helped the drug to traverse deeper in the corneal layers when compared to nanomicelle solution.

H&E staining of the whole eye (mouse model) (A) KCS control (B) after treatment with Nanoformulation, showing improvement in thickness of limbal and central epithelium and stromal regions.

Corneal penetration study in swine eyeballs. Fluorescence with color coding of depth (z in μm).

3D BIO-PRINTING

3D bio-printing technology bridges between the artificially engineered tissue construct with the versatility of native tissue by offering spatial distribution and recapitulation of personalized architectural accuracy. 3D layer by layer printing using various bio-inks can be used to design scaffolds for tumors with complex architecture and can incorporate intricate internal geometries to simulate in vivo characteristics. Patient-derived tumor xenograft cells (PDX) in 3D culture recapitulate patient-specific tumor microenvironment in vitro and generate promising outcomes in preclinical drug evaluation. In addition, co-culture of cancer-associated fibroblasts (CAFs) with tumor-specific PDX cells in appropriate 3D printing system will produce better knowledge of tumor microenvironment.