YOUNG INNOVATORS 2011
Formulation of Novel Particulate Breast Cancer
Vaccines using Spray Drying and In Vivo
Evaluation of Vaccine Efficacy
2011 AAPS Graduate Student Symposium
Awards in Biotechnology
Lipika Chablani, Suprita Tawde, Archana Akalkotkar and
Martin J. D’Souza
Department of Pharmaceutical Sciences
Mercer University, Atlanta, GA 30341
ABSTRACT
This study aims to formulate and evaluate two particulate vaccines for breast cancer using
murine breast cancer models. 67NR and 4T07 murine breast cancer cell lines were used to
prepare whole cell lysate which served as the antigen source for the particulate vaccine. 67NR
antigens were entrapped in an albumin matrix, while 4T07 antigens in a β-cyclodextrin matrix.
Formulation matrices were spray dried using Buchi 191 spray dryer to obtain vaccine
particles. The average particle size of both 4T07 β-cyclodextrin and 67NR albumin particles
was 1.3-1.7µm with slight positive surface charge. Later the vaccine efficacy was evaluated in
vivo in female Balb/c mice. Vaccine was delivered via oral, transdermal and subcutaneous
routes and serum IgG response was measured during vaccination. Both vaccines could
enhance the serum IgG levels significantly when compared to controls (p<0.001). Further the
vaccine efficacy was tested by challenging the animals with live tumor cells. Vaccinated
animals were protected from the challenge for significantly longer intervals than controls in
both particulate vaccine studies (p<0.001) indicating the vaccine was efficient in generating
protective immunity against murine breast cancer model.
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INTRODUCTION
Breast Cancer is the major female specific cancer affecting the
population in United States. Many therapies are being evaluated to
combat this cancer such as chemotherapy, surgery, hormone therapy
and radiation therapy. Most of these therapies are invasive and possess
numerous adverse effects. Immunotherapy is being explored to provide
a better treatment option to cancer patients. Various clinical trials are in
progress utilizing this approach but so far no vaccine is available in the
market. Currently our lab is investigating the efficacy of two whole cell
lysate breast cancer vaccines of two murine models via two specialized
microparticles which can be delivered orally, transdermally, and
subcutaneously in a murine model.
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MATERIALS AND METHODS
Whole cell lysate
Bio-Rad Protein Assay
67NR/4T07 cells
Challenge
Buchi 191 Spray Dryer
Balb/c mice
SEM of particles
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MATERIALS AND METHODS
Oral Route
Transdermal Route
Lectin-Nanosphere Characterization
Admin Pen
Langerhans
Cells
M-Cell
Surface
Drug Delivery Technology, Gastrointestinal Targeting, Vol. 8, No. 3
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MATERIALS AND METHODS
Immunization Scheme
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RESULTS: PARTICLE SIZE DISTRIBUTION
70
Particle %
60
67NR Albumin particles
50
4T07 B-Cyclodextrin particles
40
30
20
10
0
≤ 1µm
2µm
3µm
4µm
Particle Size
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RESULTS: PARTICLE SURFACE MORPHOLOGY
Scanning Electron Microscopy
Image of 67NR vaccine particles
Scanning Electron Microscopy
Image of 4T07 vaccine particles
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RESULTS: SDS PAGE ANALYSIS
250 kD
10 kD
Standard
67NR
Lysate
67NR
Particles in
pH 1.2
67NR
Particles in
pH 4.0
67NR
Particles in
pH 6.8
67NR
Particles in
pH 7.0
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Percentage live cells
RESULTS: MTS CELL CYTO-TOXICITY ASSAY
140
Albumin particles
120
Cyclodextrin Particles
100
80
60
40
20
0
0.0625
0.125
0.25
0.5
1
2
Negative Positive
Particle Concentration (mg/mL) Control Control
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RESULTS: IN SITU PARTICLE UPTAKE STUDY
PP = Peyer’s Patch; PPFT = Peyer’s Patch Free Tissue
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RESULTS: IN VIVO PARTICLE UPTAKE STUDY
PP = Peyer’s Patch; PPFT = Peyer’s Patch Free Tissue
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RESULTS: IN VIVO PARTICLE UPTAKE STUDY
PP = Peyer’s Patch; PPFT = Peyer’s Patch Free Tissue
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RESULTS: SERUM IgG TITERS 67NR VACCINE
1600
Control
Concentration (ng/mL)
1400
1200
1000
800
Oral Vaccine
Transdermal Vaccine
Subcutaneous Vaccine
600
400
200
0
0
4
8
Number of Doses
10
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RESULTS: TUMOR MEASUREMENTS 67NR VACCINE
600
Oral Vaccine
Tumor Volume (mm³)
500
400
Transdermal vaccine
Subcutaneous Vaccine
Control
300
200
100
0
0
1
Number of Weeks
2
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RESULTS: SERUM IgG TITERS 4T07 VACCINE
450
Concentration (µg/mL)
400
Control
Oral Vaccine
350
300
250
Transdermal Vaccine
Subcutaneous
Vaccine
200
150
100
50
0
0
2
5
Number of Doses
7
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RESULTS: TUMOR MEASUREMENTS 4T07 VACCINE
900
Tumor Volume (mm³)
800
700
600
500
Control
Oral vaccine
Transdermal Vaccine
Subcutaneous Vaccine
400
300
200
100
0
1
2
Number of Weeks
3
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DISCUSSION
• Particle size of the formulation was important to achieve efficient
particle uptake.
• The formulations were efficient in sustaining the release of antigen
and provided enteric protection when given orally.
• Both 67NR and 4T07 oral particulate vaccines required multiple
boosters to obtain significant antibody titers when compared to
transdermal and subcutaneous particulate vaccines.
• Various studies are ongoing to understand the immune pathway
followed by these vaccine particles. Flow cytometry based studies
indicate role of cellular and humoral immune response that
protected vaccinated animals longer than their control counterparts.
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CONCLUSION
• The novel approach to formulate particulate breast cancer vaccines which
can be efficiently delivered through oral, transdermal and/or subcutaneous
routes proves to be promising mode of immunization against breast cancer
as proven by two individual in vivo studies in this paper.
• This mode of immunization can be used as an individualized therapy,
where tumor cells can be isolated from the patient and cell homogenate can
be formulated into a particulate vaccine to avoid relapse and enhance
immune response.
• Nanotechnology thus can serve as a savior for millions of patients suffering
from this dreadful form of cancer.
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ACKNOWLEDGMENTS
• Dr. Martin J. D’Souza
• Grant funded by Georgia
Cancer Coalition
• AAPS-Biotechnology section
• Dr. Fred Miller
• Dr. Ray Green
• Past & current lab members
• Family and friends
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REFERENCES
•
Vyas, S. P. & Gupta, P. N. Implication of nanoparticles/microparticles in mucosal
vaccine delivery. Expert Rev Vaccines. 2007; 6: 401-18.
•
Xiang, S. D., Scholzen, A., Minigo, G., David, C., Apostolopoulos, V., Mottram, P. L.
and Plebanski, M. Pathogen recognition and development of particulate
vaccines: does size matter? Methods. 2006; 40: 1-9.
•
Anoa G., Esquisabel A., Pastor M., Talavera A., Cedréa B.,Fernándeza S.,
Sifontesa S., Arangurena Y., Faleroa G., García L. and Solís R. L., Pedrazb J. L. A
new oral vaccine candidate based on the microencapsulation by spray drying
of inactivated Vibrio cholera. Vaccine. 2011; 29: 5758-5764.
•
Lai, Y. H. & D’Souza, M. J. Formulation and evaluation of an oral melanoma
vaccine. J Microencapsul. 2007; 24: 235-52.
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CONTACT INFO
Lipika Chablani (PhD. Candidate)
Department of Pharmaceutical Sciences
College of Pharmacy and Health Sciences
3001 Mercer University Dr.
Mercer University
Atlanta, Georgia 30341
Tel.: (678)-547-6285
Fax: (678)-547-6423
Email: lipika.chablani@live.mercer.edu
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