1983022-7951 BIRLA INSTITUTE OF TECHNOLOGY

1983022-7951
BIRLA INSTITUTE OF TECHNOLOGY & SCIENCE, PILANI
HYDERABAD CAMPUS
March 2018
Final report
Study in advance topics
BITS G513
Topic of the study: liposomal formulations in cancer therapy.

Aim of the study: To know the formulation perspectives and the challenges in the formulation and search literature to gather information.

Submitted by: Vibha Deshpande
2017H1460147H
Under the supervision of
Dr swati biswas
Evaluation:
Remark:
ACKNOWLEDGEMENTI would like to express my special thanks to Dr. Swati biswas, who gave me the opportunity to study on the topic of liposomal formulation in cancer therapy.

Contents
TOC o “1-3” h z u Basic structure of liposomes PAGEREF _Toc512526270 h 4Advantages of liposomes PAGEREF _Toc512526271 h 4Disadvantages PAGEREF _Toc512526272 h 4Classification of liposomes PAGEREF _Toc512526273 h 5Materials used in preparation of liposomes PAGEREF _Toc512526274 h 6General method PAGEREF _Toc512526275 h 8Lipid film hydration by hand shaking method PAGEREF _Toc512526276 h 8French pressure cell liposomes: PAGEREF _Toc512526277 h 8Characterization of liposomes: PAGEREF _Toc512526278 h 10Uses of liposomes PAGEREF _Toc512526279 h 11APPLICATION OF LIPOSOMES PAGEREF _Toc512526280 h 11In vitro release of TAIII from LP and CD44-LP PAGEREF _Toc512526281 h 13In vivo pk studies PAGEREF _Toc512526282 h 14Invitro cytotoxicity study PAGEREF _Toc512526283 h 15References: PAGEREF _Toc512526284 h 19
Introduction
Liposomes are concentric bilayered vesicles in which an aqueous core is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids.

Liposomes were first produced in England in 1961 by Alec D. Bangham, who was studying phospholipids and blood clotting.
The size of a liposome ranges from some 20 nm up to several micrometers.

The lipid molecules are usually phospholipids- amphipathic moieties with a hydrophilic head group and two hydrophobic tails.
On addition of excess water, such lipidic moieties spontaneously originate to give the most thermodynamically stable conformation.
In which polar head groups face outwards into the aqueous medium, and the lipidic chains turns inwards to avoid the water phase, giving rise to double layer or bilayer lamellar structures.

Basic structure of liposomes
Advantages of liposomesNon ionic
Can carry both water and lipid soluble drugs
Biodegradable drugs can be stabilized from oxidation
Improve protein stabilization
Controlled hydration
Provide sustained release
Targeted drug delivery or site specific drug delivery
Stabilization of entrapped drug from hostile environment
Alter pharmacokinetics and pharmacodynamics of drugs
DisadvantagesLess stability
Low solubility
Short half life
Phospholipids undergoes oxidation, hydrolysis
Leakage and fusion
High production cost
Quick uptake by cells of R.E.S
Allergic reactions may occur to liposomal constituents
FORMATION OF LIPOSOME
When phospholipids are dispersed in water, they spontaneously form closed structure with internal aqueous environment bounded by phospholipid bilayer membranes, this vesicular system is called liposome.

Classification of liposomes
Based on structural parameters: MLV, OLV, UV, SUV ,MUV ,LUV ,GUV ,MV.
Based on method of liposome preparation: REV, MLV-REV , SPLV , FATMLV , VET , DRV.

Based on the composition and application: CL, RSVE, LCL , pH sensitive liposome, cationic liposome , immuno- liposomes
Based on structural parameters
MLV : Multilamellar large vesicles- >0.5 ?m
GUV : Giant unilamellar vesicles->1 ?m LUV
Large unilamellar vesicles->100nm
MUV : Medium sized unilamellar vesicles
SUV : Small unilamellar vesicles-20-100nm
UV : Unilamellar vesicles (all in size)
OLV : Oligolamellar vesicles- 0.1-1 ?m
MV : Multivesicular vesicles->1 ?m
Based on the composition and application
Conventional liposome : Neutral or negatively charged Phospholipid
Fusogenic liposome : Reconstitute sendai virus envelop
Cationic liposome : Cationic lipid Long circulatory liposome
Neutral high Transition temperature liposome
pH sensitive liposome : Phospholipid like Phosphatidyl ethanolamine
Immuno liposome : Long circulatory liposome with attached monoclonal antibody
Based on method of liposome preparation
REV : Single or oligolamellar vesicles made by reverse-phase evaporation method
MLV-REV : Multilamellar vesicles made by reverse phase evaporation method
SPLV : Stable plurilamellar vesicles
FATMLV : Frozen and thawed
MLV VET : Vesicles prepared by extrusion technique
DRV : Dehydration-rehydration method
Based on the composition and application
Conventional liposome : Neutral or negatively charged Phospholipid
Fusogenic liposome : Reconstitute sendai virus envelop
Cationic liposome : Cationic lipid Long circulatory liposome
Neutral high Transition temperature liposome
pH sensitive liposome : Phospholipid like Phosphatidyl ethanolamine
Immuno liposome : Long circulatory liposome with attached monoclonal antibody
Materials used in preparation of liposomesPhospholipids :
It is the major component of the biological membrane.

Two types of phospholipids are used natural and synthetic phospholipids.

The most common natural phospholipid is the phosphatidylcholine (PC) is the amphipathic molecule and also known as lecithin.

It is originated from animal (hen egg) and vegetable (soya bean).

Example: phosphodiglycerides , sphingolipids
The lipid bi-layer used in the liposomes are usually made of phospholipids and cholesterol.

Following are the
A) Naturally occurring phospholipids used in liposomes are:
Phosphatidylcholine (PC),
Phosphatidylethanolamine (PE),
Phosphatidylserine (PS).
B) Synthetic phospholipids used in the liposomes are:
Dioleoyl phosphatidylcholine (DOPC),
Distearoyl phosphatidylcholine (DSPC),
Dioleoyl phosphatidylethanolamine (DOPE),
Distearoyl phosphatidylethanolamine (DSPE).
Steroids
Cholesterol is generally used steroid in the formulation of liposomes.

It improves the fluidity of the bilayer membrane and reduces the permeability of bilayer membrane in the presence of biological fluids such as blood / plasma.
Cholesterol appears to reduce the interactions with blood proteins
Method of preparation of liposomes

General method
There are four basic methods of physical/mechanical dispersion :
Hand shaken method.
Non shaking method.
Pro – liposomes .
Freeze drying .
Lipid film hydration by hand shaking methodLipids form stacks of film from organic solution (FE/HS)
Then film is treated with aqueous medium
Upon hydration lipids swell and peel out from RB flask
vesiculate to form Multi lamellar vesicles(MLVs)
Pro-liposome
To increase the surface area of dried lipid film & to facilitate instantaneous hydration
Lipid dried over finely divided particulate matter like Nacl/sorbitol gives pro liposomes
Processing of the lipids hydrated by physical means or the mechanical treatments of MLVs :
French pressure cell liposomes:Extrusion of preformed large liposomes in french press under very high pressure .
uni or oligo lamellar liposomes of intermediate size (30-80nm ) .
Advantages
Less leakage and more stable liposomes are formed compared to sonicated forms
Vesicles prepared by extrusion technique
The size of liposomes is reduced by gently passing them through polycarbonate membrane filter of defined pore size at lower pressure

Dried reconstituted vesicles& freeze thaw sonication method

pH induced vesiculation
The transient change in pH brings about an increase in surface charge of the lipid bilayer which induces spontaneous vesiculation .
Solvent dispersion methods:

Ammonium sulphate gradient method

Characterization of liposomes:Physical characterisation
Vesicles size/shape/morphology
? Surface -charge/electrical potential
? Phase behaviour/ lamellarity
? Drug release
? % capture /free drug
CHEMICAL CHARACTERISATION
? Phospholipids /lipid concentration
? Drug concentration
? PH / Osmomolality
?Antioxidant degradation
? Phospholipids / cholesterols – peroxidation/oxidation/hydrolysis
BIOLOGICAL CHARACTERISATION
? Sterility
? Pyrogenisity
? Animal toxicity
?Plasma Stability
Uses of liposomesIn gene delivery
As drug delivery carriers.
Enzyme replacement therapy.
Chelation therapy for treatment of heavy metal poisoning.
Liposomes in antiviral/anti microbial therapy.
In multi drug resistance.
In tumour therapy.
In immunology.
In cosmetology
APPLICATION OF LIPOSOMESLiposomes in tumour therapy
Carrier of small cytotoxic molecules
Vehicle for macromolecules as cytokines genes
Liposomes in gene delivery
Gene and antisense therapy
Genetic vaccination
Liposomes in immunology
Immunoadjuvant
Immunomodulator
Liposomes as artificial blood surrogates
Liposomes as radiopharmaceutical and radio diagnostic carriers
Liposomes in cosmetics and dermatology
Liposomes in enzyme immobilization and bioreactor technology
Preclinical study: Antibody-modified liposomes for tumor-targeting delivery of timosaponin aIII
Introduction
Timosaponin AIII (TAIII), as a steroid saponin in Anemarrhena asphodeloides, has favorable potential as an antitumor candidate. However, its hydrophobicity and low bioavailability severely limit its in vivo antitumor efficacy.
TAIII also exhibits highly potent cytotoxicity against various cancer cells, such as HeLa cells,breast carcinoma cells, human colon cancer cells,human hepatocellular carcinoma (HCC) cells,PANC-1 cells, melanoma cells,and A549 human non-small-cell lung cancer cells. TAIII is nontoxic to nontransformed cells.

The mechanisms underlying the antitumor effects of TAIII involve the inhibition of tumor migration and invasion,activation of autophagy,and induction of apoptosis.

Conventional liposomes have exhibited high plasma Cl because of rapid uptake and can be mostly captured and eliminated by the phagocytic cells of the reticuloendothelial system (RES).

The incorporation of phospholipids with grafted polyethylene glycol (PEG) side chains into the membrane surface aids the liposomes to circulate longer in the bloodstream, thus evading the RES,presenting passive targeting activity by accumulating in tumors through the enhanced permeability and retention (EPR) effect.

CD44 is an extracellular protein on the cell membrane involved in tumor invasion and metastasis.

Anti-CD44 antibody, which can recognize and bind specifically to CD44 receptors, is a potent tumor-targeting ligand for improving the liposome accumulation at tumor sites and thus for enhancing cancer therapies.

CD44 receptors are overexpressed in HCC and anti-CD44 antibody-conjugated drugs have shown promising results in human clinical trials,thus, the anti-CD44 antibody ligand receptor system is a promising strategy for HCC therapy.

The cellular uptake of CD44-LP was then investigated in comparison with that of LP containing rhodamine through confocal laser scanning microscopy (CLSM).

Moreover, the in vivo tumor accumulation profile of indocyanine green (ICG)-loaded CD44-LP and LP was investigated using an IVIS imaging system.

Furthermore, using the mouse-bearing HCC cell line HepG2, the therapeutic efficacies of CD44-LP were compared with those of LP and free TAIII.

In vitro release of TAIII from LP and CD44-LPThe release of TAIII from LP and CD44-LP was investigated through a dialysis method Briefly, 1 mL of LP and CD44-LP were loaded into a dialysis bag .
The dialysis bags were immersed into release medium (PBS containing 0.5% v:v Tween 80) and agitated with a magnetic stirrer at 37°C for 48 h.

Because of its poor solubility, the concentration of TAIII in the release medium was lower than the detection limits.

Thus, liposomes in the dialysis bag were measured through HPLC–ELSD at a predetermined time
Results:
The in vitro release of TAIII from LP and CD44-LP is illustrated in Figure 2B. After the 48 h incubation period, LP and CD44-LP showed an ~55.2 and 44.7% cumulative release, respectively, significantly lower than that of free TAIII (80.8%).
This was attributable to the liposomal bilayer and outer surface with hydrophilic polymers, which delay release by forming a protective shell.

Although both the LP and CD44-LP showed favorable sustained-release capability in vitro, the release from the CD44-LP was relatively slower, possibly because of the steric protection effect endowed by anti-CD44 antibody on the surface of liposomes

In vivo pk studiesFigure 2C and Table 2, respectively, illustrate the concentration–time curve of TAIII in plasma and pharmacokinetic parameters of TAIII after intravenous injection of TAIII, LP, and CD44-LP in rats. Compared with the LP and CD44-LP, free TAIII was cleared more rapidly, as indicated by the plasma TAIII levels reaching lower than detectable limits in the TAIII
Both the LP and CD44-LP could prolong the circulation time of TAIII in blood, thus providing significantly higher AUCs than TAIII alone.

The higher AUC and mean residence time (MRT) values for LP and CD44-LP can contribute to higher plasma exposure of TAIII and greater antitumor efficiency
Notably, the TAIII volume of distribution (V) delivered by LP and CD44-LP also increased ~5.81 times and 4.47 times, compared with free TAIII

Invitro cytotoxicity study24HR 48HR
TAIII 20.41±0.80 11.74±0.50
LP 11.67±0.50 7.91±0.34
CD44- LP 14.05±0.61 5.87±0.567.

The results suggest that TAIII could be efficiently released from the LP and CD44-LP in HepG2 cells, and liposome encapsulation of TAIII could effectively enhance cytotoxicity, presumably attributable to the enhanced cellular uptake of liposomes through nonspecific endocytosis and active transport
INVITRO CELLULAR UPTAKE
HepG2 cells treated with CD44-LP showed stronger red fluorescence than those treated with nontargeted LP, and the fluorescence of both groups greatly increased as incubation time increased to 4 h.

The results revealed that CD44-targeted liposomes could effectively enhance cellular uptake in comparison with nontargeted liposomes.
By contrast, negligible rhodamine fluorescence was observed in cells pretreated with anti-CD44 antibody, possibly because anti-CD44 antibodies blocked CD44 receptors and competed with CD44-LP in receptor binding.

These results demonstrate that CD44-targeted liposomes most likely entered cells through receptor-mediated endocytosis, resulting in a higher cellular uptake

INVIVO IR IMAGING AND BIODISTRIBUTION

INVIVO ANTI-TUMOR ACTIVITY
In vivo antitumor study was evaluated on HepG2 tumor bearing mice models through intraperitoneal injection with 7.5 mg/kg physiological saline, TAIII, LP, or CD4 4-LP. Mice treated with PBS were used as the control

H$E Staining images

H&E staining images of tumors and other main organs from different treatment groups.

Tumors in the saline group were observed with larger, irregularly shaped nuclei, with some even being binucleolate, indicating cell proliferation in the tumors.

By contrast, the tumors treated with the CD44-LP showed scattered nuclei, obvious nuclear shrinkage, and cytoplasmic vacuolation, thus indicating tumor tissue necrosis.

The LP treatment group showed scattered nuclei and nuclear chromatin condensation, along with a lower inhibitory effect on tumor cell growth compared with the CD44-LP.

Furthermore, free TAIII treatment led to relatively lower tumor necrosis signals.

In addition, none of the treatment groups exhibited significant toxicity in the healthy organs compared with the control group

Conclusion
CD44-LP and LP significantly enhanced plasma half-life, reduced systemic Cl, and improved bioavailability of TAIII.

High selectivity of the CD44-LP toward CD44-positive tumor cells contributed to a more substantial uptake by HepG2 cells and a higher tumor accumulation in HepG2 xenograft mice compared with the LP, resulting in slower HepG2 xenograft growth and tumor necrosis.

In particular, CD44-LP exhibited considerably higher cytotoxicity than did LP, with a lower IC50 (48 h). CD44-LP exhibited stronger tumor inhibition; the tumor inhibitory effect was 1.3-fold higher than that of the LP.
Therefore, CD44-LP represents a promising drug delivery system with favorable biocompatibility and antitumor efficiency against CD44-positive tumor cells

References:lu lu1 Yue Ding2 Yong Zhang2 rodney JY ho3 Yuan Zhao4 Tong Zhang1 Chunrong Guo2 (n.d.) ‘Antibody-modified liposomes for tumor-targeting delivery of timosaponin aIII’, International Journal of Nanomedicine, (), pp. .

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