1. ©Immunotrex Biologics 2014 CONFIDENTIAL 1 PEG Nanoparticles: Production and Drug Delivery Structure and Function Principles of the NanoKit: A Laboratory Educational Tool Syed K. Hasan M.D. Ph.D CEO/President Arthur Watterson, Ph.D. Professor Emeritus, Organic Chemistry University of Massachusetts, Lowell Inventor & Scientific Advisor

2. Nanoparticles: An overview There are many types of nanoparticles. –Liposomes –Dendrimers –Quantum dots Nanoparticle production. –Multiple production methods –Variable stability and utility The applications for nanoparticles –Drug delivery –Molecular targeting –Visualization of biological processes ©Immunotrex Biologics 2014 CONFIDENTIAL 2

3. Other Circulating Delivery Systems and Agents ©Immunotrex Biologics 2014 CONFIDENTIAL 3 Delivery SystemStrengthsDrawbacks Liposomes  Biocompatible  Clinical success in cancer (breast, ovarian, lymphoma), antifungals  Often too large  Stability and sterility issues  Poor encapsulation Dendrimer  High surface units per area  Adaptable interior  Difficult synthesis – time consuming, expensive  Scalability issues Imaging Agent (Magnetic, QDots)  Easily modifiable  Powerful MR, fluorescence contrast agents  Less suitable for drug delivery applications  Potential toxicity (QDots)

4. Flexible Chemo-Enzymatic Synthetic Design: An Example + Enzyme Polymerization Hydrophilic (water loving) group PEG monomer Tri-functional Linker ©Immunotrex Biologics 2014 CONFIDENTIAL 4 Step 1: Polymer Formation Hydrophilic PEG polymer Polyethylene Glycol (PEG) monomer and the tri-functional linker are mixed and subject to enzymatic polymerization. The resultant polymer is linear hydrophilic PEG polymer, with an “open” linker.

5. ©Immunotrex Biologics 2014 CONFIDENTIAL 5 Flexible Chemo-Enzymatic Synthetic Design: An Example Step 2: Hydrophobic Group Attachment Hydrophilic PEG polymer with Hydrophobic Functional Groups Hydrophobic group (water hating) Chemical Attachment + Hydrophilic PEG polymer (water loving) (n)(n) Can be CH3, OH, COOH, NH2 Hydrophilic PEG polymer is chemically attached to the hydrophobic side chain group at the “open” 3 rd linker. The resultant polymer is linear hydrophilic PEG polymer, with an attached side chain.

6. ©Immunotrex Biologics 2014 CONFIDENTIAL 6 Formation of Nanospheres Add water !! PEG Surface Hydrophobic core Self-Assembly Step 3: Self-Assembly in Water Amphiphilic PEG Copolymer Nanosphere w/ Functional Groups Linear hydrophilic PEG polymer is exposed to water and self-assembles. The resultant Amphiphilic PEG Copolymer Nanosphere is complete and ready to go !

7. Atomic force microscopy of PEG 1500, c-8 side chain nanoparticles in aqueous solution with nanoparticles absorbed on a freshly cleaved mica surface. (t=1.5 hrs.) “On the Cover” Horizontal distance: 19.6 nm ©Immunotrex Biologics 2014 CONFIDENTIAL 7

8. Enzyme Catalyzed Synthesis of Pegylated Copolymer ©Immunotrex Biologics 2014 CONFIDENTIAL 8 Copolymer was synthesized by green chemistry approach using Candida Antarctica Lipase (CAL) enzymatic catalysis Simple Apparatus & Process

9. Structure:  The balance of hydrophobic and hydrophilic forces within a molecule is important.  The Properties of a molecule can change dramatically with changes in structure.  The formation of nanoparticles can be a relatively simple process. Function:  Encapsulation of a target molecule is dependent upon the structure of the polymer and the drug.  Rate of release is influenced by the molecular structure of the encapsulated drug. ©Immunotrex Biologics 2014 CONFIDENTIAL 9 Principles of the NanoKit

10.  Polymer A has a ten carbon side chain  Polymer B has a four carbon side chain  Polymer A will encapsulate drug  Polymer B will not encapsulate drug  Polymer B side chains are less hydrophobic than those of polymer A:  Nanoparticle micelle will form with polymer A and not form with polymer B.  The rate of drug release from a nanoparticle delivery system is dependent upon the structure of the nanoparticle and the drug. ©Immunotrex Biologics 2014 CONFIDENTIAL 10 Key Teaching Points:

11. NanoKit contains:  Two vials of two different polymers (A & B)  Vials of drug (Aspirin, Acetaminophen, Naproxen)  Step-by-step experimental protocol Laboratory requirements:  Solvent for mixing  Evaporation equipment  Filtration equipment  Weighing balance ©Immunotrex Biologics 2014 CONFIDENTIAL 11

12. Extensions of the NanoKit experimental protocol:  UV analysis of the materials  NMR analysis of products  Comparison of different drugs The NanoKit may be used at multiple chemistry course levels. ©Immunotrex Biologics 2014 CONFIDENTIAL 12

13. Thank you for the opportunity to discuss the NanoKit. Immunotrex Biologics is committed to developing advanced technology educational kits in support of its biotechnology and molecular medicine platforms. ©Immunotrex Biologics 2014 CONFIDENTIAL 13 Questions ?