Investigation of the Stability and Cellular Uptake of Self-Associated

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Investigation of the Stability and Cellular Uptake of Self-Associated Monoclonal Antibody (MAb) Nanoparticles by Non-small Lung Cancer Cells. Asha R. Srinivasan, Ashakumary Lakshmikuttyamma, and Sunday A Shoyele Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp3005935 • Publication Date (Web): 05 Aug 2013 Downloaded from http://pubs.acs.org on August 7, 2013

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Molecular Pharmaceutics

Investigation of the Stability and Cellular Uptake of Self-Associated Monoclonal Antibody (MAb) Nanoparticles by Non-small Lung Cancer Cells. Srinivasan, Asha R., Lakshmikuttyamma, Ashakumary, Shoyele, Sunday A.* Department of Pharmaceutical Sciences, School of Pharmacy, Thomas Jefferson University, Philadelphia, PA. 19107. USA.

*Sunday A. Shoyele, PhD Department of Pharmaceutical Sciences School of Pharmacy Thomas Jefferson University 901 Walnut Street, Philadelphia, PA 19107 Tel: 215-503-3407 Fax: 215-503-9052 Email: [email protected]

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Table of Content Graphics

FITC

DAPI

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Abstract The inability to deliver MAbs to intracellular target still remains a limitation to their application in cancer therapy and diagnosis. Selective targeting of MAbs to oncoproteins in cancer cells while avoiding their accumulation in normal cells may reduce some of the well documented adverse effects accompanying antibody therapy. One of the remarkable characteristics of malignant cells is the alteration in the biological properties of the cellular plasma membrane. Taking advantage of this alteration, we hope to selectively deliver self-associated MAb nanoparticles to cancer cells while reducing their accumulation in normal cells. We hypothesized that self-associated MAb nanoparticles can be preferentially taken up by non-small lung cancer cells in comparison to normal cells due to the absence or dysfunction of tight junctions (TJ) in confluent cancer cells and increased permeability of the cancer cell membrane. Self-associated bevacizumab nanoparticles were prepared and characterized for particle size and biochemical stability. Fluorescence microscopy, TEM and flow cytometry revealed that these bevacizumab nanoparticles were internalized by A549 cells three times more than MRC-5 Cells. Macropinocytosis and energy-dependent pathways were elucidated to be involved in their uptake by A549 cells. Further, uptake was by non-specific interaction with cell membrane. Results obtained from this study suggest that self-associated MAb nanoparticles can be selectively delivered to cancers cell. Key words: monoclonal antibodies; nanoparticles; oncoproteins; non-small cell lung cancer; endocytosis.


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Introduction Monoclonal antibodies (MAbs) are the most widely used form of cancer immunotherapy. Nevertheless, their effectiveness as research tools and therapeutic agents would be substantially enhanced if they had the ability to act intracellularly1. However, antibodies are often viewed as too large to access intracellular locations2, 3. This assumed limitation of MAb therapeutics informs the restriction of targets to those located at the surface or exterior of host cells4, 5. Recent findings have however, shown that MAbs can indeed cross cell membranes to access intracellular targets3, 6. This is particularly significant considering that many oncogenic proteins such as vascular endothelial growth factor (VEGF) which is localized in vesicular organelles, and intracellular phosphatases / kinases and transcription factors localized either in the cytosol or nucleus are located within the cell 7-9. Hence, intracellular oncoproteins present a compelling target for MAb therapy. Compared to normal cells, the absence or dysfunction of tight junctions (TJ) in confluent cancer cells, and the marked amounts of intracellular materials released from cancer cells into surrounding fluid, provide corroborative evidence of increased permeability of the cancer cell membrane10-13. To this end, we hypothesize that: self-associated MAb nanoparticles will be substantially internalized by cancer cells in comparison to normal cells. To test this hypothesis, self-associated bevacizumab nanoparticles were prepared by a recently reported nanoprecipitation method14. Bevacizumab a humanized monoclonal antibody against vascular endothelial growth factor (VEGF) has shown encouraging signs in the treatment of non-small cell lung



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cancer (NSCLC) when used alone or in combination with chemotherapy15-17. It gained FDA approval in 2006 for use with paclitaxel and carboplatin as first-line treatment for those with advanced NSCLC. Cancer cells tend to overexpress VEGF, a potent stimulator of angiogenesis, facilitating cancer growth and metastasis17-18. Internalization of bevacizumab into cancer cells is highly important as previous reports have shown that the intracellular pool of VEGF could be responsible for resistance to bevacizumab in cancer therapy8, 9, 19-20. To this end, intracellular VEGF provides a compelling target for MAbs in cancer therapy. The internalization of self-associated bevacizumab nanoparticles in NSCLC cell line (A549) was investigated in comparison to normal lung epithelial cells (MRC-5). Further, the internalization pathways of these self-associated bevacizumab nanoparticles were elucidated using transmission electron microscopy (TEM), fluorescence microscopy and flow cytometry. Retained anti-VEGF activity of the bevacizumab nanoparticles was investigated using human umbilical vein endothelial cells (HUVEC) while antiproliferative activity against NSCLC was investigated using A549 cell line. This study provides the first evidence that self-associated MAb nanoparticles can be selectively delivered to cancer cells. Experimental section Materials Cell lines and culture conditions. The NSCLC line A549, human umbilical vein endothelial cells (HUVEC) and normal lung fibroblast cell line MRC-5 were obtained from the American Type Culture Collection, Rockville, MD. A549 cells were maintained in F12K medium supplemented with 10% fetal bovine serum along with 1% antibiotics in


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a humidified air atmosphere with 5% carbon dioxide. HUVECs were maintained in endothelial basal medium supplemented with endothelial cell growth kit-VEGF in a humidified air atmosphere with 5% carbon dioxide. MRC-5 was maintained in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum in humidified air atmosphere with 5% carbon dioxide. Chemicals Bevacizumab was kindly provided by Genetech Inc., South San Francisco, CA. Dialysis was performed on the bevacizumab using a 100kDa dialysis tubes in order to remove any excipients from the bevacizumab sample. The bevacizumab was then lyophilized and stored at -20°C for further use. Lyophilized bevacizumab is labeled as unprocessed bevacizumab particles for the purpose of this study. Polysorbate 80 (Polyoxyethylene (80) sorbitan monooleate), polysorbate 20 (Polyoxyethylene (20) sorbitan monolaurate), and brij 97 (polyethylene glycol monooleyl ether) were supplied by Sigma-Aldrich, Saint Louis, MO. All other excipients and reagents were of reagent grades and were purchased from Fisher Scientific, Pittsburgh, PA. Methods Production of self-associated bevacizumab nanoparticles. Nanoparticles were produced by dissolving 5 mg/ml of excipient-free lyophilized bevacizumab particles in 0.01N HCl containing different concentrations of polysorbate 80 (tween 80), polysorbate 20 (tween 20) or brij 97. The mixture was then slowly titrated with 0.1N NaOH to bring the pH of the mixture to 8.4 which is the iso-electric point of bevacizumab while continuously mixing on a magnetic stirrer21. At the iso-electric point, bevacizumab nanoparticles were spontaneously precipitated. The colloidal suspension



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was then centrifuged using a microcentrifuge at 6500 RPM for 5 minutes. The supernatant was decanted and the pellet formed rinsed with double distilled de-ionized water. Dry nanoparticles were prepared by re-suspending the nanoparticles in water by vortexing. The suspended particles were then snap-frozen using liquid nitrogen before being loaded into freeze dryer (Labconco Freezone 4.6, Missouri). Lyophilization was performed for 24 hours. Percentage Yield of Nanoparticles The percentage yield of nanoparticles produced from the nanoprecipitation process was determined by taking samples from the supernatant following centrifugation and analyzing for protein content using UV absorption at 280 nm. % yield was calculated as: Total amount of bevacizumab – unprecipitated bevacizumab / Total amount of bevacizumab X 100

Photon correlation Spectroscopy (PCS) Particle size (by intensity) and zeta potential measurements were performed by PCS using Zetasizer Nano ZS (Malvern Instruments, UK). The pellets formed after centrifugation at 6500 RPM for 5 minutes were thoroughly rinsed and resuspended in deionized water by vortexing. The samples were then sonicated for approximately 5 minutes. Intensity autocorrelation was measured at a scattering angle (θ) of 173 degrees at 25°C. The Z-average and polydispersity index (PDI) were recorded in triplicate. For zeta potential measurement, the samples were loaded into a universal dip cell (Malvern Instruments, UK) before recording the zeta potential in triplicate.


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Scanning Electron Microscopy (SEM) The morphology of the bevacizumab nanoparticles was observed by scanning electron microscopy using the Zeiss Supra 50VP system (Zeiss, Germany). Powders were mounted onto aluminum stubs using double sided adhesive tape and were made electrically conductive by coating in a thin layer of gold. The coated samples were then examined under microscope operated at an acceleration voltage of 5 kV. Far-UV Circular Dichroism (CD) CD measurements were performed with Jasco J-810 Spectropolarimeter (Jasco, MD, USA) operating at 20°C using 0.5 mg/ml of reconstituted solutions of bevacizumab in acetate buffer. CD spectra were obtained in the far UV region (260-190 nm) using a quartz cell of 0.1 cm path length in order to probe the stability of the secondary structure of the manufactured nanoparticles. A scanning speed of 50 nm/min with a 0.5-second response time was applied followed by five accumulations for each sample. Relevant blank spectra were subtracted from sample spectra to get the net spectra. The experiment was repeated in triplicate for each sample. Size Exclusion-HPLC (SE-HPLC) SE-HPLC was performed using an Alliance HPLC System; Waters 2695 separation module (Waters, MA, USA) combined with a Waters 2998 Photo-diode Array Detector. A TSK Gel 3000 SWXL column (300 mm x 7.8 mm) (Tosoh Cooperation, OH, USA) was used. 20 µl of the reconstituted bevacizumab, dissolved in 0.1 M acetate buffer (pH 5) at 200 µg/ml was injected using phosphate buffered saline (PBS) pH 7.4 as the mobile



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phase. The separation was performed at a flow rate of 0.5 ml/min. UV detection was performed at 214 nm. Chromatograms were recorded using the Empower Pro® software.

Retained In vitro Anti-VEGF Activity The in vitro anti-VEGF activity assay was adapted from a previously published method22. Briefly, HUVECs grown at 80% confluence were harvested and seeded in 2 x 96 well plates at 2 x 105 cells/well in ice-cold endothelial basal growth medium (serum free) with no growth factors and FBS supplementation. 50 µL of a wide concentration range of rhVEGF (0-1000 ng/mL) was added into designated wells in four replicates. Cells in complete growth medium were used as positive control to assess HUVEC proliferation. For bevacizumab inhibition, different concentrations of bevacizumab (0-500 ng/mL) were mixed with 50 ng/mL of rhVEGF and incubated at 37ºC in a humidified air atmosphere with 5% carbon dioxide for 2 hours prior to adding the cell suspension. The plate was continuously incubated for 4 days. At the end of incubation, 25 µL of alamarBlue (Sigma Aldrich, MO) was added to each well and incubated for additional 6 hours under same conditions. The plate was then read at 530/590 nm excitation/ emission on a fluorescence plate reader. The alamarBlue dye is a fluorometric growth indicator based on metabolic activity, which is reflective of extent of cellular proliferation. The control sample contained cells deprived with FBS and growth factors for 3 days. A nonspecific human IgG was also tested to exclude any possibility of a non-specific effect mediated by IgG. A control sample consisting of surfactants dissolved in the growth medium was also tested to exclude any anti-VEGF effect from the surfactants.


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Cytotoxicity Assay Cytotoxic effect of bevacizumab was assessed in adenocarcinoma cells (A549) using MTT assay. Cells (1x104 cells / well) were seeded in 96 well plates and incubated at 37ºC in a humidified air atmosphere with 5% carbon dioxide for 48 hours. The cells were treated with varying concentrations of unprocessed bevacizumab particles and reconstituted bevacizumab nanoparticles (0-1000 µg/mL) and incubated for 72 hours. At the end of incubation (after 72 hrs), MTT reagent was added to each well and incubated for 3 hours. The media was aspirated and 100 µL DMSO was added. The plate was read at 560 nm. Cells treated with DMSO and relevant surfactants were used as a control. Fluorescence Microscopy A549 and MRC-5 cells (2x104 cells/ well) were seeded in 8 well coated glass slides and incubated for 48 hours. PBS washed cells were incubated with FITC-labeled bevacizumab particles suspended in the serum free medium (100 µg/ml) for 60 minutes. Cells were washed with PBS, fixed with 2% paraformaldehyde and incubated at room temperature for 20 minutes. PBS washed cells were then blocked with 5% BSA for 30 min at room temperature. Cells were stained with AlexaFluro-555 labeled wheat germ agglutinin (WGA) and 4', 6-diamidino-2-phenylindole (DAPI) to visualize Plasma membrane and nucleus respectively. Cells were mounted and observed under Leica DMI 6000B fluorescence microscope (Leica Microsystems, PA). Uptake Experiments A549 and MRC-5 cells were used to investigate the uptake of self-associated bevacizumab nanoparticles. About 1 million cells/ well were seeded in a 12 well plate



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and incubated for 48 hours. Cells were then treated with 100 µg/ml of the FITC conjugated nanoparticles resuspended in serum-free medium and incubated for 60 minutes. The cells were trypsinized, centrifuged at 300x g for 5 minutes and the pellet was washed and resuspended in 0.4% trypan blue (TB) solution in PBS to quench the extracellular FITC fluorescence. TB, while quenching the FITC fluorescence of noninternalized particles, causes them to fluoresce red whereas an internalized particle will fluoresce green23. Cells were then centrifuged; the TB solution was removed before the cell pellets were resuspended in PBS. The samples were then analyzed by flow cytometry (BDFACS caliber). 10,000 cells were measured in each sample. To elucidate whether the uptake of bevacizumab nanoparticles was due to specific interaction with the cell membrane or not, A549 cells incubated in medium containing 10% FBS was treated with 100000 µg/ml of unlabeled bevacizumab 60 minutes prior to the treatment with 100 µg/ml of FITC-labeled bavacizumab nanoparticles. Mean Fluorescence Intensity obtained from this experiment was compared to that obtained when the A549 cell were treated with 100 µg/ml of FITC-labeled bavacizumab nanoparticles alone. For the elucidation of the mechanisms of internalization, cells were pre-incubated for 60 minutes at 37ºC/ 5% carbon dioxide with 2 µg/mL nocodazole, 0.1% sodium azide/50 mM deoxy glucose, 75 µM dynasore and 2µg/mL filipin before being treated with the nanoparticles. Transmission Electron Microscopy (TEM) Visual observation of particle internalization and translocation was gained using TEM. Approximately 5x105 cells (A549) were seeded in 60 mm2 polystyrene dishes for 48


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hours. PBS washed cells were treated with 100 µg/mL bevacizumab nanoparticles and incubated at the same time. However, they were fixed at different time points – 15 minutes, 60 minutes and 4 hours. At the end of each time point, the cells were fixed in 2% gluteraldehyde with 1% tannic acid in 0.05M phosphate buffer. The cells were rinsed three times in 0.1 M phosphate buffer. The cells were post fixed in 2% osmium tetroxide in 0.1 M phosphate buffer. After rinsing them is deionized water the cells were stained with 1% uranyl acetate in deionized water. Cells were spun down in warm agarose and the cell pellet was dehydrated in graded steps of acetone and infiltrated with Spurr's embedding media. The blocks were polymerized at 65ºC in a convection oven. The resulting blocks were cut with a Diatome diamond knife on a Leica Ultracut UCT microtome. The thin sections were picked up with copper grids and observed in a FEI Tecnai 12 TEM. Electron micrographs were captured with an AMT XR111 11 megapixel CCD camera. Statistical Analysis Results are expressed as mean ± standard deviation, unless otherwise indicated. Statistical significance difference between two groups was determined by two-tailed Student’s t test. A p-value of 0.05 was taken as statistically significant. Results Particle Characterization The percentage yield of bevacizumab nanoparticles as determined by UV absorption at 280 nm was found to vary between 90 -103%. The size, zeta potential and morphology of the self-associated bevacizumab nanoparticles were measured by PCS (Zetasizer) and SEM. Table 1 shows the influence of non-ionic



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surfactants on the particle size and charge density on the surface of self-associated bevacizumab nanoparticles. The surfactants seemed to have a concentration-dependent influence on the size and zeta potential of the nanoparticles. The size of the unprocessed bevacizumab particles could not be measured using photon correlation spectroscopy (PCS) because the instrument can only be used for particles between 0.3nm to 10 µm. According to the Manufacturer’s Manual, this is because PCS is based on Brownian motion and particles with size above 10 µm tend to quickly sediment. In this situation, any size recorded by the instrument would only be due to artifact. However, the size of the unprocessed bevacizumab as estimated from the SEM measurement was approximately 20 µm. For surfactant controls, different concentrations of the surfactants used in the nanoprecipitation process were dissolved in 0.01 N HCl and titrated with 0.1N NaOH up to pH 8.4. The zeta potential of the control surfactants was measured and the result is presented in Table 2. In case of nanoparticles, an increase in size was observed as the concentration of the non-ionic surfactant in the precipitation medium increased. For instance, bevacizumab nanoparticles precipitated from a precipitation medium containing 0.1%w/v, 0.2% and 0.3% tween 80 measured 395.4 ± 2.2, 552.8 ± 0.8 and 668.4 ± 1.1 nm respectively. The same trend was observed with the zeta potential measurement. SEM micrograph in Figure 1A revealed that the unprocessed bevacizumab (Lyophilized bevacizumab from the supplied solution after dialysis to remove any excipients in the solution) was plate-like and irregularly shaped. However, the surfactant-free bevacizumab nanoparticles (Figure 1B) were spherical but agglomerated. 0.1% tween 80containing bevacizumab nanoparticles (Figure 1C) appeared similar to the nanoparticles


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containing 0.1%w/v tween 20 (Figure 1D). However, 0.1%w/v brij 97-containing nanoparticles appeared sponge-like in shape (Figure 1E).

Table 1. Particle size, PDI and zeta potential of bevacizumab nanoparticles as measured by photon correlation spectroscopy (PCS). Sample

Particle Diameter (nm)*

PDI*

Zeta* potential (mV)

Particles precipitated with 0.1% Tween 80

395.4±2.2

0.2±0.0

-13.7±0.7


552.8±0.8

0.1±0.0

-18.1±0.8


668.4±1.1

0.2±0.0

-25.6±1.5


334.4±2.6

0.2±0.0

-10.5±0.5


530.9±5.9

0.2±0.0

-12.0±0.1


634.9±9.5

0.2±0.0

-13.5±0.3

Particles precipitated with 0.1% Brij 97

365.6±4.1

0.1±0.1

-9.7±0.4


596.5±1.5

0.1±0.0

-11.7±0.6


774.2±0.9

0.1±0.0

-13.3±0.2

Particles precipitated without surfactant (negative control)

239.2±1.0

0.1±0.0

-5.1±0.7

*Data presented as Mean ± SD.

Table 2. Zeta potential of surfactant controls measured by PCS Surfactant Control Zeta Potential (mV) 0.1% tween 80 -12.9 ± 0.8 0.2% tween 80 -13.1± 1.2 0.3% tween 80 -13.4 ± 1.5



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Figure 1. SEM micrographs of bevacizumab particles. A, Lyophilized bevacizumab from the supplied solution after dialysis to remove any excipients in the solution (scale bar, 20µm). B, surfactant-free bevacizumab nanoparticles (scale bar, 200 nm). C, 0.1% tween 80 bevacizumab nanoparticle (scale bar, 200 nm). D, 0.1% tween 20 bevacizumab nanoparticles (scale bar, 200 nm). E, 0.1% brij 97 bevacizumab nanoparticles (scale bar, 200 nm)

Far-UV CD Spectroscopy The effects of the non-ionic surfactants: tween 80, tween 20 and brij 97 on the retention of the secondary structure of the bevacizumab following the nanoprecipitation process and subsequent reconstitution in acetate buffer at pH 5 were investigated using far-UV


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CD spectroscopy. Figure 2 reveals that the far-UV CD spectrum of the unprocessed bevacizumab was characterized by a negative maximum at a wavelength of 218 nm and a positive maximum at 202 nm. These are characteristics typical for antibodies because of their high β-sheet content24-26. Following reconstitution, all the tween 80-containing bevacizumab nanoparticles retained their beta-sheet secondary structure. However, the spectrum of the surfactant-free bevacizumab nanoparticles showed perturbations in the secondary structure as shown in Figure 2. Far-UV CD spectra obtained from tween 20 and brij 97-containing bevacizumab nanoparticles were consistent with that of the reconstituted tween 80-containing bevacizumab nanoparticles.

Figure 2. Far-UV CD spectra obtained from reconstituted (in 0.1M acetate buffer, pH 5) bevacizumab nanoparticles.



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SE-HPLC The presence of aggregates and fragments in the reconstituted bevacizumab nanoparticles was investigated using SE-HPLC. Figure 3 reveals the chromatograms obtained from the SE-HPLC analysis. The main peaks at a retention time of approximately 13 minutes represent the intact bevacizumab monomer. The minor peaks at approximately 11 minutes suggest the presence of dimers in the reconstituted unprocessed bevacizumab while the peaks present at approximately 14 and 15 minutes represent the presence of fragments. Peaks from all the reconstituted nanoparticles were similar to those seen in the unprocessed bevacizumab. The percentage monomer, aggregates and fragments recovered from each nanoparticle formulation following reconstitution was determined and compared to that of the unprocessed bevacizumab. Table 3 reveals that the unprocessed bevacizumab contained 94.9 ± 0.4% monomers following reconstitution. Reconstituted surfactant-containing bevacizumab nanoparticles contained similar amounts of monomers to that of the unprocessed bevacizumab suggesting that the bevacizumab content of the surfactant-containing nanoparticles was mainly the intact monomers following reconstitution at pH 5. However, surfactant-free bevacizumab contained 14% fragments suggesting degradation in the bevacizumab in the surfactantfree nanoparticles.


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Figure 3. An overlay of the chromatograms gained from SE-HPLC analysis of different bevacizumab nanoparticles following reconstitution in 0.1M acetate buffer (pH 5) Table 3. SE-HPLC analysis of reconstituted bevacizumab nanoparticles showing monomer, aggregate and fragment contents. Sample

% Monomer*

% dimer*

%HMW*

% Fragment*

% Relative recoverya

Unprocessed bevacizumab Surfactant-free nanoparticles Particles precipitated with 0.1% Tween 80 Particles precipitated with 0.2% Tween 80 Particles precipitated with 0.3% Tween 80 Particles precipitated with 0.1% Tween 20 Particles precipitated with 0.2% Tween 20 Particles precipitated with 0.3% Tween 20 Particles precipitated with 0.1% Brij 97 Particles precipitated with 0.3% Brij 97

94.9 ± 0.4

0.0

0.6

4.5 ± 0.1

100.0 ± 0.0

85.2 ± 0.4

0.8 ± 0.0

0.0

14.0 ± 0.4

110.6 ± 1.9

93.9 ± 0.1

0.0

0.0

6.0 ± 0.1

103.6 ± 1.5

94.6 ± 0.2

0.0

0.0

5.5 ± 0.2

112.9 ± 2.0

93.6 ± 0.1

0.0

0.0

6.4 ± 0.1

98.3 ± 1.4

93.7 ± 0.5

0.0

0.0

6.3 ± 0.2

107.1 ± 1.5

92.7 ± 0.6

0.0

0.0

7.3 ± 0.4

106.0 ± 3.8

91.9 ± 0.6

0.0

0.0

8.2 ± 0.5

105.0 ± 0.5

94.8 ± 0.9

0.0

0.0

5.1 ± 1.0

116.7 ± 6.1

93.0 ± 1.0

0.0

0.0

7.0 ± 1.0

109.1 ± 1.8

* Data presented as Mean ± SD, n = 3 a Relative recovery = area under the curve for dissolved nanoparticle / area under the curve for the unprocessed.



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Retained In vitro Anti-VEGF Activity Cell survival was measured in HUVEC treated with rhVEGF. AlamarBlue assay showed a VEGF induced concentration dependent cell survival in HUVECs (Fig. 4). The VEGF treated HUVECs were used to study the inhibitory effect of bevacizumab. Significant decrease in cell survival was observed in all samples treated with different forms bevacizumab compared to samples treated with IgG (Fig 5). Reconstituted tween 80containing bevacizumab nanoparticle formulations displayed similar cytotoxicity to that of the unprocessed bevacizumab particles. This result indicates that the reconstituted bevacizumab nanoparticles exhibit the same biological property as unprocessed bevacizumab. However, the surfactant-free bevacizumab nanoparticles did not show similar effect suggesting loss of stability without the surfactant acting as a stabilizer. Students T test showed no significant difference between the activity of the surfactantcontaining nanoparticles and the unprocessed bevacizumab. However, the effect of surfactant-free nanoparticles was significantly different (P < 0.05) from the unprocessed bevacizumab particles at all concentrations. The controls (human IgG1 and tween 80) produced no anti-VEGF activity. However, the activity of all the bevacizumab samples was significantly different (**P

Investigation of the Stability and Cellular Uptake of Self-Associated

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