ProFECT Technology

Novel Proprietary Lipid Nanoparticle Formulation for Gene and Nucleic Acids Delivery Used in Therapeutics and Vaccines

ProFECT
 
ProFECT is a liposome assembly technique to enhance nucleic acids cargo loading into PEGylated neutral liposomes for improved biocompatibility and pharmacokinetics. This results in liposomal nanocarriers with efficient nucleic acid encapsulation and enhanced cellular association, while minimizing the cytotoxic effects associated with cationic-mediated transfection. ProFECT is a quick, easy, reproducible, scalable method for nanoparticle-mediated applied transfection. T&T Scientific’s ProFECT™ Transfection is a proprietary and patent-protected lipid-based transfection system designed for safe and effective nucleic acid delivery in a broad range of cell types with high viability and low lipid-associated toxicity. ProFECT™, a natural lipid composition comprised of non-cationic phospholipid and sterol components, is modified with functional amino acids for highly efficient encapsulation of nucleic acids and enhanced intracellular delivery. This easy-to-use lipid formulation is intended for preclinical in vitro studies aimed at translational development of nucleic acids therapeutics and vaccines.

  • Problem and Solution
  • Components
  • Why ProFECT?
  • Data and Validations
  • Instruction

Problem and Solution

Problem:

A major challenge in nanomedicine is the translation from laboratory formulation to large scale production and has limited clinical success in the field of nano-therapeutics. While translational success is limited with conventional drug delivery systems, the development of safe and efficacious nanocarriers is crucial to improving the bench-to-bedside success.

Solution: ProFECT Plus

ProFECT is a lipid nanoparticle assembly technique to enhance siRNA cargo loading into PEGylated neutral liposomes for improved biocompatibility and pharmacokinetics. This results in lipid nanoparticle nanocarriers with efficient nucleic acid encapsulation and enhanced cellular association, while minimizing the cytotoxic effects associated with cationic-mediated transfection. ProFECT is a quick, easy, reproducible, scalable method for nanoparticle-mediated applied transfection.

  • Composite lipid film 
(1mg per rxn)
  • Lipid-based nucleic acid condenser (1 vial per rxn)
  • Injection buffer (EtOH based buffer, 5ml per 10 rxn)
  • Condensing buffer (Tris-HCl based buffer, 10ml per 10 rxn)
  • Highly Translational: Comprised of natural lipid components
  • Improved cellular association
  • Low Toxicity: Eradicates toxicity due to charged liposomes
  • Easy to Use Protocol: One-step assembly
  • Enhanced encapsulation efficiency of the siRNA load
  • Cost-effective
  • Reduced nucleic acid leakage

Gene therapy has the potential to attenuate a variety of diseases by altering the molecular pathways leading to pathogenesis. However, the issue of delivery remains a primary hurdle in the successful translation of gene therapeutics. Cationic liposomes are beneficial experimental gene vectors due to electrostatic-mediated packaging of nucleic acids. However, the potential for clinical translation is limited in that cationic liposomes are associated with with reduced biocompatibility and poor pharmacokinetics. PEGylated neutral liposomes (PLPs), comprised of naturally occurring neutral lipids and surface grafted polyethylene glycol (PEG), are considerably more advantageous in terms of biocompatibility and increased drug stability. However, the addition of the PEG layer reduces the cell-associative properties of PLPs. Cell-penetrating peptides can be grafted onto the surface of liposomes to enhance cell delivery in order to overcome this so-called “PEG Dilemma”, but assembly must be controlled to prevent CPP-induced drug leakage.

Enhanced EE% of siRNA in ProFECT LNP

Encapsulation efficiency of nearly 100% can be achieved with ProFECT LNP formulation. Increasing lipid:siRNA (wt-to-wt) increases siRNA encapsulation efficiency EE%.

Fisher, R. K., Mattern-Schain, S. I., Best, M. D., Kirkpatrick, S. S., Freeman, M. B., Grandas, O. H., & Mountain, D. J. (2017). Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications. Journal of Surgical Research, 219, 136-144.

 Enhanced Stability of ProFECT-siRNA Complex

ProFECT LNP provides total protection of siRNA cargo. Gel exclusion assay confirms siRNA load is stable when exposed to RNase digestion.

 

Fisher, R. K., Mattern-Schain, S. I., Best, M. D., Kirkpatrick, S. S., Freeman, M. B., Grandas, O. H., & Mountain, D. J. (2017). Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications. Journal of Surgical Research, 219, 136-144.

STEM Images of ProFECT LNP Morphology

Scanning Transmission Electron Microscopy (STEM) image of extruded ProFECT LNP. STEM images verify morphology and nanoscale size (<100nm) of ProFECT LNP product.

Enhanced Cell Association of ProFECT LNP

Scanning Transmission Electron Microscopy (STEM) image of extruded ProFECT LNP. STEM images verify morphology and nanoscale size (<100nm) of ProFECT LNP product.

Fisher, R. K., Mattern-Schain, S. I., Best, M. D., Kirkpatrick, S. S., Freeman, M. B., Grandas, O. H., & Mountain, D. J. (2017). Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications. Journal of Surgical Research, 219, 136-144.

Enhanced Cell Association of ProFECT LNP

ProFECT exhibits superior cellular delivery compared to PEGylated formulations. Fluorescence microscopy images (Tx Red) of human aortic smooth muscle cells (HASMC) demonstrating cell-associative properties of ProFECT LNP formulation (0.5 mol % Rhodamine-PE).

Fisher, R. K., Mattern-Schain, S. I., Best, M. D., Kirkpatrick, S. S., Freeman, M. B., Grandas, O. H., & Mountain, D. J. (2017). Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications. Journal of Surgical Research, 219, 136-144.

Reduced Cytotoxicity of ProFECT vs. Cationic LNP

Cytotoxicity of ProFECT demonstrates reduced cytotoxicity compared to PEGylated cationic LNP formulation. Live/Dead stain of HASMC showing lower cytotoxicity of ProFECT LNP compared to PEGylated cationic LNP.

Fisher, R. K., Mattern-Schain, S. I., Best, M. D., Kirkpatrick, S. S., Freeman, M. B., Grandas, O. H., & Mountain, D. J. (2017). Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications. Journal of Surgical Research, 219, 136-144.

 ProFECT Significantly Silences GAPDH Expression

Gene silencing of ProFECT is superior to PEGylated cationic LNP formulation. At 200 and 400 nM GAPDH siRNA, ProFECT transfection reagent provides more effective gene knockdown relative to nontransfected cells than PEGylated cationic LNP (DOTAP:Chol).

Fisher, R. K., Mattern-Schain, S. I., Best, M. D., Kirkpatrick, S. S., Freeman, M. B., Grandas, O. H., & Mountain, D. J. (2017). Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications. Journal of Surgical Research, 219, 136-144.