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					Biodegradable Nanoparticles for Cancer
               Therapy


        Jamboor K. Vishwanatha, Ph.D.
        Dean and Professor
        Graduate School of Biomedical Sciences
Poly D,L lactide-co-glycolide (PLGA)

    Extensively investigated polyester
    Numerous assets
        Release profile can be controlled
        Nanoparticle size can be controlled
        Capable of the capture of any therapeutic agent
             Hydrophobic (ATRA, doxorubicin, 5 fluorouracil)
             Hydrophilic (DNA, protein, small molecules)
        Potential for development of targeted or
         combinational therapies
        Very low immunogenicity and cytotoxicity
        High transfection potential
           Properties of PLGA

Hydroxyl                                                       Carboxyl
terminus                                                       terminus


                      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
    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
Nanotechnology Applications

     Gene Delivery

     Chemotherapeutic Delivery
Gene Delivery: Formulation of DNA
      loaded nanoparticles

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

   Our formulation parameters include the use of a non-
    solvent.
   The addition of a non-solvent accomplishes several
    goals
      Minimization of shear forces
      Decreases in particle size
What do these particles look
           like?
                          A                             B                        C




A                                                              C



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.
              PLGA 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 Uptake




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

                                                                        110




                                                 Percent viable cells
                                                                        100
                                                                         90
                                                                         80
                                                                         70
                                                                         60
                                                                         50
                                                                         40
                                                                         30
                                                                         20
                                                                         10
                                                                          0
                                                                              0   250   500   750   1000   1250
    A                                            BB                                 Dose in micrograms

  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.


We have also observed no cytotoxic effects on cells at concentrations up to
3 mg/mL
     pDrive-sh AnxA2 loaded nanoparticles
      can serve to mediate prostate cancer
               cellular migration
                  A                     B                    C

      24 hours




                   D                    E                     F

      48 hours




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

                          150
                                                                          DU-145 cells were exposed to
                                                                          nanoparticles over an 8 day time
Number of Cells (X1000)




                                                                          course. Control cell growth is
                          100                                             indicated by the solid black line.
                                                                          Unloaded blank nanoparticles
                          50
                                                                          (dashed line) display no effect
                                                                          upon cell growth or growth rate.
                                                                          pDrive-sh AnxA2 loaded
                           0                                              nanoparticles (dotted line)
                                0   1   2   3      4 5 6      7   8   9
                                                Time (days)
                                                                          significantly diminish cellular
                                                                          growth and rate of growth.
                                 In vivo analysis of nanoparticle efficacy


A                                A                            B                                               C


                                  Control HBSS                pDrive-sh AnxA2                             Blank unloaded
                                  treated:                    nanoparticle treated:                       nanoparticle
                                                                                                          treated:
                                  18 days                     27 days
                                                                                                              9 days
    B1250                                                                        C                      110
Tumor volume (mm^3)




                                                               Control                                  100                                    Control




                                                                                     Percent survival
                      1000                                     pDrive-sh AnxA2                           90                                    pDrive-sh AnxA2
                                                               Blank                                     80                                    Blank
                                                                                                         70
                      750
                                                                                                         60
                                                                                                         50
                      500                                                                                40
                                                                                                         30
                      250                                                                                20
                                                                                                         10
                        0                                                                                 0
                             0   10   20      30    40   50                                                   0   10   20      30    40   50
                                      Time (days)                                                                      Time (days)
                                                    In vivo continued

                                                     Tumor progresison in male nude mice

                                        1600
                                                                   R2 = 0.9742
                                        1400

                  Tumor volume (mm^3)   1200                                                sh
                                        1000                                                con
                                                                   R2 = 0.9574
                                                                                            blnk
                                        800
                                                                                            Linear (blnk)
                                        600                                                 Linear (con)
                                                                           R2 = 0.9138      Linear (sh)
                                        400

                                        200

                                          0
                                               0       5     10      15      20      25
                                                             Time (days)




Control 21 days                                    Sh treated 27 days                     Blank 9 days
        Chemotherapeutic Delivery:
            ‘Nanocurcumin’
   Polymeric nanoparticles encapsulating
    Curcumin (anti-cancer drug)

   Curcumin:
     Diferuloylmethane, a yellow polyphenol
      extracted from Curcurma longa
     Therapeutic agent in traditional Indian
      medicine
     Curcumin Vs Nanocurcumin
    Free Curcumin            Nanocurcumin


   Poorly dispersible      Dispersible in water
    in water                Sustained drug release kinetics
                            Improved Bioavailability
   Reduced
                            Improved cellular uptake
    Bioavailability
                            Improved inhibition of
                             clonogenicity of cancer cell
                             lines
             Characterization

   Percent Yield : 90-94

   Encapsulation Efficiency: > 95%
Particle Size Analysis:
        Formulation Optimization

Batch    PVA    Sonication   Particle Size   Percent   Percent
        Conc.     Time          range         Yield  Encapsulation

 A      1.5%    1.0 min      150-250 nm      90.00      93.73

 B      1.5%    2.0 min      100-200 nm      92.78      94.60
 C      2.0%    2.0 min      20-100 nm       92.01      90.88
Surface Morphology:




                            500nm


   Transmission Electron 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-vitro Release Kinetics
                         75
    % Cuucumin release
                         60

                         45

                         30

                         15

                          0
                              0   50   100      150   200   250
                                         Time (h)



 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
                                             Cell Viability Assay
                         1.2                                                                        1.2
Cell viability (Rel)




                                                                    Cell viability (Rel)
                         0.8                                                                        0.8


                         0.4                                                                        0.4
                                   LNCaP                                                                          PC3
                          0                                                                          0
                               0     10        20     30       40                                         0         10          20       30    40
                                      Curcumin (mM)                                                                      Curcumin (mM)




                                                                                                                                                Control
                                                                                                                                                Blank
                                                                                                                                                Free curcumin
                         1.2                                                                         1.2                                        Nano curcumin




                                                                             Cell viability (Rel)
  Cell viability (Rel)




                         0.8                                                                         0.8


                         0.4                                                                         0.4
                                   PWR1E                                                                          SKBr3
                          0                                                                               0
                               0     10        20         30   40                                             0            20             40
                                          Curcumin (mM)                                                             Curcumin (mM)
Second generation nanoparticles
    We are working on the development of
     targeted nanotherapeutics
    The goal of our work is to deliver locally
     higher concentrations of drug to diseased
     cells or tissues
    These nanoparticles are capable of selective
     attachment of nucleophilic substrates
        Antibodies
        Proteins (Transferrin)
        Peptides (NLS sequences)
        Small molecules (N-acetyl cysteine)
Schematic diagram for the development of
         targeted nanoparticles




    Using a platform technology we first generate an activated
     nanoparticle
    In a second reaction the targeting agent is conjugated to the
     outer surface of the nanoparticle
                       Mode of action




   The targeted nanoparticle finds the specific cellular target
   The nanoparticle binds to the surface of the cell
      If the target is internalized (i.e. folate receptors) the
       nanoparticle is carried to the intracellular environment
      If the target is not internalized (i.e. annexin A2) the delivery
       system has been engineered to release the nanoparticle at
       the surface of the cell allowing for endocytosis to occur
PSMA targeting under co-culture
         conditions


                    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 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 eye




Intravitreal
injection




                Potential convective current for vitreous
Nanoparticles are capable of reaching the retinal
                   cell 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
                            mL. 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-
         vitreal injection of nanoparticles
                                                         The ciliary body is
                                                          located adjacent to the
                                                          lens in the anterior
                                                          portion of the eye
                                                         One of the functions is
                                                          the production of vitreal
                                                          fluid
                                                         It may be possible to use
                                                          accumulation in the ciliary
                                                          body as a drug reservoir
Confocal image of the ciliary body from pig retinal
sections. It appears that a higher accumulation of        for sustained release
nanoparticles is occurring.
                                                         Issues of drug transport
                                                          to the retina still remain
Reduction of reactive oxygen species in
        various disease states
   We are developing multi-phase nanoparticles for
    protection against oxidative damage to cells.

       We expect these nanoparticles to provide an
        immediate scavenging response to cellular oxidative
        stressors (first phase).
       In the second phase we are going to provide
        sustained long-term protection against oxidative
        damage.
       We anticipate applications of these nanoparticles in
        the areas of glaucoma, ischemic recovery (stroke
        victims) and COPD.
       Preliminary data suggest that we are able to reduce
        the effective dose of a known protective agent by 25
        fold.
     Protection of retinal ganglion cells from
                 reactive oxygen
                                      IAA is an chemical
                                       inducer of reactive
                                       oxygen. Treatment
                                       was with 8 mM
                                      N-acetyl cysteine was
                                       administered at a
                                       concentration of 5 mM
Control             IAA
                                      N-acetyl cysteine was
                                       conjugated to the
                                       surface of the
                                       nanoparticle at a
                                       concentration of 0.5
                                       mM
                                      Visualization was
N-acetyl cysteine   Nanoparticle       performed 20 hours
                                       after IAA induction
Acknowledgements:
        Dr. Arthur Braden
        Dr. Anindita Mukerjee
        Mallika Valapala

      jvishwan@hsc.unt.edu

				
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