This document provides an overview of the evolution of protein and macromolecule delivery systems. It discusses techniques used for stability testing, bioassay, UV spectroscopy, Bradford assay, differential scanning calorimetry, chromatography, and electrophoresis. Recent advances include smart polymer-based delivery systems using temperature sensitive, phase sensitive, pH sensitive, and photosensitive polymers. Hybrid protein delivery systems are also discussed, including ligated gene fusion and synthetically linked hybrid conjugates.

1. Guide by,
Dr. Sanjeevni R. Desai
Prepared by,
M r. Dnyaneshwar B. Ningule
(First year M.Pharmcy Roll no.10)
EVOLUTION OF DELIVERY SYSTEM OF
PROTEIN AND OTHER MACROMOLECULES
Department of Pharmaceutics
Vasantidevi Patil Institute of Pharmacy , Kodoli

2. SRNO INDEX
1 Introduction
2 Protein & Peptide
3 Stability Testing
4 Bioassay
5 UV Spectroscopy
6 Barford assay
7 Difrantial scanning calorimetery
8 Cromatography
9 Electrophoresis
10 Recent Advancement
11 Hybrid Protein Delivery System

3. INTRODUCTION
Protein and Peptide drug delivery system are the Novel drug
Delivery System. Proteins and peptides are the most abundant
components of biological cells. They exist functioning such as
enzymes, hormones, structural element and immunoglobulin

4. PROTEIN & PEPTIDE
PROTEINS :- Proteins are the large organic compounds
made of amino acids arranged in a linear chain and joined
together by peptide bonds,
a protein > 50 amino acids
PEPTIDES :-These are short polymers formed from the
linking, in a defined order of amino acids.
• peptide < 50 amino acids

5. • EVOLUTION OF DELIVERY SYSTEM
OF PROTEIN AND OTHER
MACROMOLECULES INCLUDES
FOLLOWING TECHNIQES.
Stability Testing
Bioassay
UV Spectroscopy
Barford assay
Difrantial scanning calorimetery
Cromatography
Electrophoresisi

6. • BIOASSAY
• Due to complexity of proteins, bioassay are required to
assess
potency of the formulation. Bioassay are of two types : in
vitro
and in vivo. In case of in vitro bioassays response of cells to
hormones and growth factors is monitored. In case of in
vivo
bioassay pharmacological response of animals to proteins is
monitored. Eg: post injection BSL in rabbits is measured for
bioassay of insulin

7. • UV SPECTROSCOPY
• Proteins containing aromatic amino acid residues such
as phenyl alanine, tyrosine, tryptophan can be detected
by UV spectroscopy. It can be used as an analytical
determination method

8. • BRADFORD ASSAY
• This assay employs the principle that in the presence of
proteins in an acidic medium, absorption maximum of
coomassie brilliant blue G-250 dye changes. If there is
no protein to bind, the solution remains brown. The dye
forms a complex with carboxyl end of proteins by Van
der Waals forces to form a blue colored solution. The
intensity of the colored solution can be measured using
a spectrophotometer to detem1ine the concentration of
the protein in the sample.

9. • DIFFERENTIAL SCANNING
CALORIMETRY
Used as a tool for inves6gating transitiOns of
conformation as a function of temperature and, more
importantly, the effect of potential stabilizing
excipients in a protein solution.

10. • CHROMATOGRAPHY
• To study stability of proteins and peptides.
Various
modes used are
1. Normal Phase HPLC
2. Reverse Phase HPLC
3. Ion Exchange
4. Chromatofocusing

11. • ELECTROPHORESIS
• Sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) used. Proteins are
denatured by boiling in the SDS solution. All
charges of protein are masked by negative charge of
dodecyl sulphate. Thus protein moves on
polyacrylamide gel strictly on basis of size of
protein molecule. This technique is useful for
determining molecular weight of proteins. For
visualization of proteins on the gel, reagents used
are silver nitrate, coomassie brilliant blue dye

12. RECENT ADVANCES
• Smart polymer based delivety system:
• biodegradable polymeric systems delivering protein and
peptide drugs
• smart polymer-based injectable drug delivery systems
• Smart polymers are classified according to the external
stimulus they respond to:
1. Temperature sens1t1ve:
Poly (ethylene oxide )-poly (propylene oxide )-poly( eth
ylene oxide) triblock copolymers
2. Phase sensitive polymers: Poly (D,L-lactide), Poly (D
,L-lactide-co glycolide)
3. pH sensitive polymers: Poly(methacrylic acid g-ethyle
ne glycol
4. Photosensitive polymers: polyvinyl alcohol

13. • HYBRID PROTEIN DELIVERY
SYSTEMS :-
• Heterologous hybrid proteins can be designed
bearin
g the combined or reordered features of one or more
proteins that display effector functions, protection a
bilities and recognition properties.
• Site specific hybrid proteins may be produced:
1. Ligated gene fusion hybrid delivery systems: Hybr
id protein between interferon y and TNF p have be
en developed.
2. Synthetically linked hybrid conjugates

14. • REFERENCE :-