Biodegradable Nanoparticles for Cancer Therapy

Jamboor K. Vishwanatha, Ph.D.Jamboor K. Vishwanatha, Ph.D.Dean and Professor Dean and Professor Graduate School of Biomedical SciencesGraduate School of Biomedical Sciences

Poly D,L lactide-co-glycolide (PLGA)Poly D,L lactide-co-glycolide (PLGA)

Extensively investigated polyesterExtensively investigated polyester Numerous assetsNumerous assets

Release profile can be controlledRelease profile can be controlled Nanoparticle size can be controlledNanoparticle size can be controlled Capable of the capture of any therapeutic agentCapable of the capture of any therapeutic agent

Hydrophobic (ATRA, doxorubicin, 5 fluorouracil)Hydrophobic (ATRA, doxorubicin, 5 fluorouracil) Hydrophilic (DNA, protein, small molecules)Hydrophilic (DNA, protein, small molecules)

Potential for development of targeted or Potential for development of targeted or combinational therapiescombinational therapies

Very low immunogenicity and cytotoxicityVery low immunogenicity and cytotoxicity High transfection potentialHigh transfection potential

Lactide Glycolide

PLGA undergoes acid catalyzed hydrolysis to release cellular metabolites of lactic and glycolic acid

PLGA nanoparticle size can be controlled through variations in nanoparticle formulation conditions

Hydroxyl terminus

Carboxyl terminus

Variations in lactic acid to glycolic acid ratios effect the degradation profile of the polymer (release rate)

Degradation rate is also affected through variations in the intrinsic viscosity (i.v.) of the polymer

Properties of PLGA

Nanotechnology ApplicationsNanotechnology Applications

Gene DeliveryGene Delivery

Chemotherapeutic DeliveryChemotherapeutic Delivery

Gene Delivery: Formulation of DNA Gene Delivery: Formulation of DNA loaded nanoparticlesloaded nanoparticles

Traditional formulation is accomplished through the use Traditional formulation is accomplished through the use a W/O/W double emulsion solvent evaporation technique a W/O/W double emulsion solvent evaporation technique

Our formulation parameters include the use of a non-Our formulation parameters include the use of a non-solvent.solvent.

The addition of a non-solvent accomplishes several The addition of a non-solvent accomplishes several goalsgoals Minimization of shear forcesMinimization of shear forces Decreases in particle sizeDecreases in particle size

What do these particles look What do these particles look like?like?

Pictures of PLGA nanoparticles following completion of fabrication (Panel A; Size bar ~100 nm). Panel B is a TEM image PLGA nanoparticles (Size bar ~500nm). Panel C is a TEM image

of antibody targeted nanoparticles. Here the nanoparticles appear translucent with colloidal gold labeled anti-mouse antibody as the dark specks (Size bar indicates 100nm).

Nanoparticles are stable at 4oC indefinitely and easily resuspend in isotonic buffers or cell media.

A C

A B C

PLGA nanoparticle sizePLGA nanoparticle size

A B C

Figure 2: Formulation parameters and their effect on size of plasmid DNA loaded nanoparticles. Through the optimal choice of solvent/non-solvent systems we can control the size of nanoparticles produced. Panel A: Solvent: Chloroform, Non-solvent: Water; size range 100->1000 nm. Panel B: Solvent: Chloroform, Non-solvent: Ethanol; size range 100-400 nm. Panel C: Solvent: Chloroform, Non-solvent: Methanol; size range 51-138 nm.

It is important to be able to control the ultimate size of the particles in order to achieve optimal transfection of cells and cross physiological barriers (i.e. blood brain barrier and nuclear pore complexes).

Intracellular UptakeIntracellular Uptake

Nanoparticles labeled with Nile Red appear red and can be seen within the cells after 1 hour of incubation.

Nanoparticle efficiency: Uptake and Nanoparticle efficiency: Uptake and transfectiontransfection

Transfection ability and cytotoxic effects of nanoparticles. PLGA nanoparticles were dual loaded with sulforhodamine 101 (red) and GFP plasmid DNA (green) and exposed to DU-145 cells. Four days post-transfection cells were visualized under laser confocal microscopy. Greater than 90% of the cells display transcription of GFP encoding plasmid DNA and cellular uptake of the nanoparticles (panel A). Unloaded nanoparticles were evaluated for cytotoxic effects upon cells (panel B). Greater than 90 percent cell viability at the maximal dose of 1 mg/ml can be seen.

A B 0 250 500 750 1000 12500

102030405060708090

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Dose in micrograms

Percent viable cells

We have also observed no cytotoxic effects on cells at concentrations up to 3 mg/mL

B

pDrive-sh AnxA2 loaded nanoparticles pDrive-sh AnxA2 loaded nanoparticles can serve to mediate prostate cancer can serve to mediate prostate cancer

cellular migrationcellular migration

Migration of DU-145 cells upon administration of plasmid DNA loaded nanoparticles and blank unloaded nanoparticles. Transfection of DU-145 cells was performed for 4 days and visualized 24 and 48 hours after plating of the migration assay. Control cells are seen in panel A and D respectively. There is a tremendous reduction in cellular migration of DU-145 cells treated with plasmid DNA loaded nanoparticles (panel B and E). There is no effect upon migration when treated with blank unloaded nanoparticles (panel C and F). At 48 hours cells have been counter stained with crystal violet to enhance visualization.

A B C

D E F

24 hours

48 hours

pDrive-sh AnxA2 nanoparticles also pDrive-sh AnxA2 nanoparticles also effect prostate cancer cellular effect prostate cancer cellular

proliferationproliferation

0 1 2 3 4 5 6 7 8 90

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Number of Cells (X1000)

DU-145 cells were exposed to nanoparticles over an 8 day time course. Control cell growth is indicated by the solid black line. Unloaded blank nanoparticles (dashed line) display no effect upon cell growth or growth rate. pDrive-sh AnxA2 loaded nanoparticles (dotted line) significantly diminish cellular growth and rate of growth.

In vivo analysis of nanoparticle efficacy

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Tumor volume (mm^3)

A

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Percent survival

B C

pDrive-sh AnxA2 nanoparticle treated:

27 days

Control HBSS treated:

18 days

Blank unloaded nanoparticle treated:

9 days

BA C

In vivo continued

Tumor progresison in male nude mice

R2 = 0.9742

R2 = 0.9574

R2 = 0.9138

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Linear (con)

Linear (sh)

Sh treated 27 daysControl 21 days Blank 9 days

Chemotherapeutic Delivery: Chemotherapeutic Delivery: ‘Nanocurcumin’‘Nanocurcumin’

Polymeric nanoparticles encapsulating Polymeric nanoparticles encapsulating Curcumin (anti-cancer drug)Curcumin (anti-cancer drug)

Curcumin:Curcumin:Diferuloylmethane, a yellow polyphenol Diferuloylmethane, a yellow polyphenol

extracted from extracted from Curcurma longaCurcurma longaTherapeutic agent in traditional Indian Therapeutic agent in traditional Indian

medicinemedicine

Curcumin Curcumin VsVs Nanocurcumin Nanocurcumin

Free CurcuminFree Curcumin

Poorly dispersible Poorly dispersible in waterin water

Reduced Reduced BioavailabilityBioavailability

NanocurcuminNanocurcumin

Dispersible in waterDispersible in water Sustained drug release kinetics Sustained drug release kinetics Improved BioavailabilityImproved Bioavailability Improved cellular uptake Improved cellular uptake Improved inhibition of Improved inhibition of

clonogenicity of cancer cell clonogenicity of cancer cell lineslines

Characterization Characterization

Percent Yield : 90-94Percent Yield : 90-94

Encapsulation Efficiency: > 95%Encapsulation Efficiency: > 95%

Particle Size Analysis:Particle Size Analysis:

Formulation OptimizationFormulation Optimization

BatchBatch PVA PVA

Conc.Conc.

Sonication Sonication TimeTime

Particle Size Particle Size rangerange

Percent Percent YieldYield

Percent Percent EncapsulationEncapsulation

AA 1.5%1.5% 1.0 min1.0 min 150-250 nm150-250 nm 90.0090.00 93.7393.73

BB 1.5%1.5% 2.0 min2.0 min 100-200 nm100-200 nm 92.7892.78 94.6094.60

CC 2.0%2.0% 2.0 min2.0 min 20-100 nm20-100 nm 92.0192.01 90.8890.88

Surface Morphology:Surface Morphology:

500nm

Transmission Electron Microscopy

Confocal Microscopy:Confocal Microscopy:

Curcumin

PLGA Nanoparticles

Curcumin nanoparticles were observed under Confocal Microscope (Carl Zeiss LSM 410). For curcumin: λex is 450nm

and λem is 488nm

In-vitroIn-vitro Release Kinetics Release Kinetics

Curcumin nanoparticles were incubated in PBS (pH 7.4) and at different time points, the supernatant was analyzed at λ:450nm for cumulative curcumin release

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Cell Viability AssayCell Viability Assay

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PC30

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( )Cell viability Rel

SKBr30

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( )Cell viability Rel

Control

Blank

Free curcuminNano curcumin

Second generation nanoparticlesSecond generation nanoparticles

We are working on the development of targeted We are working on the development of targeted nanotherapeuticsnanotherapeutics

The goal of our work is to deliver locally higher The goal of our work is to deliver locally higher concentrations of drug to diseased cells or tissuesconcentrations of drug to diseased cells or tissues

These nanoparticles are capable of selective These nanoparticles are capable of selective attachment of nucleophilic substratesattachment of nucleophilic substrates AntibodiesAntibodies Proteins (Transferrin)Proteins (Transferrin) Peptides (NLS sequences)Peptides (NLS sequences) Small molecules (N-acetyl cysteine)Small molecules (N-acetyl cysteine)

Schematic diagram for the development of Schematic diagram for the development of targeted nanoparticlestargeted nanoparticles

Using a platform technology we first generate an activated Using a platform technology we first generate an activated nanoparticlenanoparticle

In a second reaction the targeting agent is conjugated to the In a second reaction the targeting agent is conjugated to the outer surface of the nanoparticleouter surface of the nanoparticle

Mode of actionMode of action

The targeted nanoparticle finds the specific cellular targetThe targeted nanoparticle finds the specific cellular target The nanoparticle binds to the surface of the cellThe nanoparticle binds to the surface of the cell

If the target is internalized (If the target is internalized (i.e.i.e. folate receptors) the folate receptors) the nanoparticle is carried to the intracellular environmentnanoparticle is carried to the intracellular environment

If the target is not internalized (If the target is not internalized (i.e.i.e. annexin A2) the delivery annexin A2) the delivery system has been engineered to release the nanoparticle at system has been engineered to release the nanoparticle at the surface of the cell allowing for endocytosis to occurthe surface of the cell allowing for endocytosis to occur

PSMA targeting under co-culture PSMA targeting under co-culture conditionsconditions

Activated nanoparticles loaded with sulforhodamine 101 (red) were quenched and exposed to PSMA antibody. Following 1 hour, untargeted nanoparticles were exposed to a co-culture of PC-3 and LNCaP C4-2 cells under dymanic motion conditions for 30 minutes. No preferential uptake of nanoparticles is observed.

Targeted preferential uptakeTargeted preferential uptake

PSMA targeted nanoparticles were loaded with sulforhodamine 101 (red) and exposed to a co-culture of PC-3 and LNCaP C4-2 cells for 30 minutes under dynamic motion.

Samples were fixed in paraformaldehyde and visualized through laser confocal microscopy.

PC-3 cells are shown with yellow arrows, LNCaP C4-2 cells are shown with green arrows. It is evident that there is a preferential uptake of targeted nanoparticles to the LNCaP C4-2 cell line.

Anatomy of the eyeAnatomy of the eye

Intravitreal injection

Potential convective current for vitreous

Nanoparticles are capable of reaching the retinal Nanoparticles are capable of reaching the retinal cell layerscell layers

Pig retinal section 4 days post-intra vitreal injection of nanoparticles. Nanoparticles were loaded with sulforhodamine 101 (red) and GFP plasmid DNA (green). The concentration of nanoparticles was 1 mg/75 μL. Nuclear visualization was performed using hematoxylin. The section shown is located in the posterior portion of the retina.

Ganglion cell layer

Outer Nuclear Layer

Inner Nuclear Layer

Retina

Investigation of the ciliary body after intra-Investigation of the ciliary body after intra-vitreal injection of nanoparticlesvitreal injection of nanoparticles

The ciliary body is located The ciliary body is located adjacent to the lens in the adjacent to the lens in the anterior portion of the eyeanterior portion of the eye

One of the functions is One of the functions is the production of vitreal the production of vitreal fluidfluid

It may be possible to use It may be possible to use accumulation in the ciliary accumulation in the ciliary body as a drug reservoir body as a drug reservoir for sustained releasefor sustained release

Issues of drug transport Issues of drug transport to the retina still remainto the retina still remain

Confocal image of the ciliary body from pig retinal sections. It appears that a higher accumulation of

nanoparticles is occurring.

Reduction of reactive oxygen species in Reduction of reactive oxygen species in various disease statesvarious disease states

We are developing multi-phase nanoparticles for protection We are developing multi-phase nanoparticles for protection against oxidative damage to cells.against oxidative damage to cells.

We expect these nanoparticles to provide an immediate We expect these nanoparticles to provide an immediate scavenging response to cellular oxidative stressors (first scavenging response to cellular oxidative stressors (first phase).phase).

In the second phase we are going to provide sustained long-In the second phase we are going to provide sustained long-term protection against oxidative damage.term protection against oxidative damage.

We anticipate applications of these nanoparticles in the areas We anticipate applications of these nanoparticles in the areas of glaucoma, ischemic recovery (stroke victims) and COPD.of glaucoma, ischemic recovery (stroke victims) and COPD.

Preliminary data suggest that we are able to reduce the Preliminary data suggest that we are able to reduce the effective dose of a known protective agent by 25 fold.effective dose of a known protective agent by 25 fold.

Protection of retinal ganglion cells from Protection of retinal ganglion cells from reactive oxygenreactive oxygen

IAA is an chemical IAA is an chemical inducer of reactive inducer of reactive oxygen. Treatment was oxygen. Treatment was with 8 mMwith 8 mM

N-acetyl cysteine was N-acetyl cysteine was administered at a administered at a concentration of 5 mMconcentration of 5 mM

N-acetyl cysteine was N-acetyl cysteine was conjugated to the conjugated to the surface of the surface of the nanoparticle at a nanoparticle at a concentration of 0.5 mMconcentration of 0.5 mM

Visualization was Visualization was performed 20 hours performed 20 hours after IAA inductionafter IAA induction

NanoparticleN-acetyl cysteine

Control IAA

Acknowledgements:

Dr. Arthur Braden

Dr. Anindita Mukerjee

Mallika Valapala

jvishwan@hsc.unt.edu