Background Figure 1: Dose response curves illustrating differences in the therapeutic index. A) A drug with a favorable therapeutic index does not

Transcription

1 Background Figure 1: Dose response curves illustrating differences in the therapeutic index. A) A drug with a favorable therapeutic index does not induce toxicity at doses that achieve effective tumor control. B) A drug with an unfavorable therapeutic index has a nearly 1 to 1 ratio of tumor response to normal tissue damage. (Figure Credit: Bernier, J et al. Radiation oncology: a century of achievements. Nature Reviews Cancer )

2 Background Figure 2: The tumor microenvironment affects drug delivery and efficacy. A) Tumor cells are present at great distances from blood vessels and are densely packed. B) Many tumor cells are so distant from the vasculature they occupy a hypoxic environment (green); this distance impedes effective systemic drug delivery. C) Densely packed cancer cells and the extracellular matrix decrease drug concentration at the core of the tumor. (Figure Credit: Minchinton, A. et al. Drug penetration in solid tumors. Nature Reviews Cancer )

3 Background Figure 3: The enhanced permeability and retention effect allows for the passive targeting of nanoparticles to tumors. Tumors display leaky vasculature and a disordered structure that acts as a filter for systemically administered nanoparticles, targeting them to the tumor. The lack of a functioning lymphatic system prevents drug from being removed, but elevates the pressure of the tumor, impeding passive nanoparticle accumulation. (Figure Credit: Bisht, S. et al. Dextran-doxorubicin/chitosan nanoparticles for solid tumor therapy. WIREs Nanomedicine and Nanobiotechnology )

4 A) B) Figure 1: Structure, molecular weight, and weight percent Rhodamine B of the synthesized polymers with varying compositions and zwitterionic-to-peg ratios. A) Polymers functionalized with maleimide; note that has a significantly higher weight percent Rhodamine B than the other polymers. B) Control polymers lacking maleimide; note that has a significantly lower weight percent Rhodamine B than the other polymers, and that the control polymers have generally lower weight percent Rhodamine B than the maleimide polymers.

5 A) D) Emission (RFU) Emission (RFU) Polymer Fluorimetry 488 nm Excitation; 560 nm to 595 nm Emission Emission Wavelength (nm) Polymer Fluorimetry 561 nm Excitation; 600 nm to 620 nm Emission Doxorubicin Polymer Maleimide Free Doxorubicin B) C) Total Fluorescence (RFU) Total Fluorescence (RFU) Total Polymer Fluorescence 488 nm Excitation; 560nm to 595 nm Emission Polymer Type Doxorubicin Polymer Maleimide Free Doxorubicin Doxorubicin Polymer Maleimide Free Doxorubicin E) F) Total Polymer Fluorescence 561 nm Excitation; 600 nm to 620 nm Emission Polymer Fluorescence Normalization 488 nm Excitation; 560nm to 595nm Emission Total Fluorescence 488Ex Em Normalized Fluorescence Total Fluorescence 488Ex Em Normalized Fluorescence Total Fluorescence 488Ex Em Normalized Fluorescence Total Fluorescence 488Ex Em Normalized Fluorescence Total Fluorescence 488Ex Em Normalized Fluorescence Polymer Fluorescence Normalization 561 nm Excitation; 600 nm to 620 nm Emission Total Fluorescence 561ex 610/20 Em Normalized Fluorescence Total Fluorescence 561ex 610/20 Em Normalized Fluorescence Total Fluorescence 561ex 610/20 Em Normalized Fluorescence Total Fluorescence 561ex 610/20 Em Normalized Fluorescence Wavelength (nm) Total Fluorescence 561ex 610/20 Em Normalized Fluorescence Polymer Type Figure 2: Polymer fluorescence normalization. Polymer fluorescence was determined for the excitation and emission ranges of the Amnis Flow Sight and BD FACs Aria in order to normalize fluorescence for the variance in bound Rhodamine B between the polymers. A) Polymer Fluorimetry at the excitation and emission range for the Amnis Flow Sight. B) Integration of the emission profile in A. C) Normalization to for the Amnis Flow Sight. D) Polymer Fluorimetry at the excitation and emission range for the BD FACs Aria. E) Integration of the emission profile in D. F) Normalization to for the BD FACs Aria.

6 A) B) Figure 3: Analysis of polymer toxicity in Human Bone Marrow Mesenchymal Stem Cells and Murine DO11.10 T Cells with trypan blue exclusion on a ViaCell Cell Counter (Beckman Coulter). A) The viability of Human Bone Marrow Mesenchymal Stem Cells was unaffected the maleimide polymers 20 minutes after polymer conjugation. B) The viability of DO11.10 T Cells was unaffected by polymer conjugation and maintained vaiability equal to the untreated control for 72 hours. Note that the large loss in viability observed in T Cells treated with Free Doxorubicin is not present in T Cells treated with Polymer Maleimide Doxorubicin.

7 Figure 4: Flow Cytometry fluorescence normalization. Polymer fluorescence was determined for the excitation and emission ranges of the Amnis Flow Sight and BD FACs Aria in order to normalize cellular fluorescence for the variance in bound Rhodamine B between the polymers. A) Amnis Flow Sight Median Intensity values for cellular fluorescence of the 5 maleimide rhodamine polymers with 488 nm excitation and 560 nm to 595 nm emission in hbmmscs. Values are blanked against the unstained control. B) Normalization of cellular fluorescence in A with values from Figure 3. Note that is the best binding of the maleimide polymers in hbmmscs. C) BD FACs Aria Median Intensity values for cellular fluorescence of the 5 maleimide rhodamine polymers and 5 control polymers with 561nm excitation and 600 nm to 620 nm emission in DO11.10 T Cells. Values are blanked against the unstained control. D) Normalization of cellular fluorescence in C with values from Figure 3. Note that is the best binding of the maleimide polymers in DO11.10 T Cells. Flow Cytometry Analysis of Polymer Conjugated hbmmscs 488 nm Excitation 560 nm-595 nm Emission Relative Fluorescence Units (RFU) A) B) Normalized Flow Cytometry Analysis of Polymer Conjugated hbmmscs 488 nm Excitation 560 nm-595 nm Emission Relative Fluorescence Units (RFU) Polymer Type Polymer Type Normalized Flow Cytometry Analysis of Polymer Conjugated DO11.10 T Cells 561 nm Excitation 600 nm- 620 nm Emission Relative Fluorescence Units (RFU) C) D) Polymer Type Flow Cytometry Analysis of Polymer Conjugated DO11.10 T Cells 561 nm Excitation 600 nm- 620 nm Emission Relative Fluorescence Units (RFU) Polymer Type

8 Overlay Polymer (590/50) Differential Interference Contrast Figure 5: Polymer trafficking of in hbmmscs. The subcellular location of polymer binding resembles the string like structure of the mitochondria. All polymers were internalized, and polymer trafficking is also unaffected by polymer composition.

9 Figure 6: Colocalization of with MitoTracker Deep Red. fluorescence (Pseudocolored red) appears to perfectly overlap with the fluorescence of MitoTracker Deep Red (Pseudocolored blue). Note the similar string like structures observed by both stains, as well as the degree of overlap, as seen by purple in the overlay.

10 Figure 7: Quantitative colocalization of with MitoTracker Deep Red. A region of interest for colocalization quantification was drawn in order to eliminate background from colocalization calculations (left image). and MitoTracker Deep Red were confirmed to colocalize quantitatively, with a Pearson s coefficient of 0.79 and a Mander s overlap of 0.84 (right image). Colocalizing pixels are highlighted in red in the left image.

11 Overlay Polymer (590/50) Differential Interference Contrast Figure 8: localization in hbmmscs prior to treatment with the mitochondrial membrane depolarizer FCCP. Polymer 2 Maleimide remains in the mitochondria as confirmed by previous colocalization experiments (see Figures 6 and 7).

12 Overlay Polymer (590/50) Differential Interference Contrast Figure 9: mitochondrial localization in hbmmscs is lost immediately after treatment with the mitochondrial membrane depolarizer FCCP. does not display the characteristic mitochondrial staining pattern as the pre-fccp image (Figure 8). In addition, the polymer fluorescence is dispersed and has lost intensity. Notably, MitoTracker Deep Red responded in the same way (data not shown).

13 Overlay Doxorubicin (590/50) Differential Interference Contrast Figure 10: The Doxorubicin Maleimide Polymer reveals a similar staining pattern in hbmmscs as the other rhodamine polymers and appears to be trafficked to the mitochondria. Notably, fluorescence from the Doxorubicin polymer is not seen in the nucleus.

14 Free Doxorubicin (10 um Weight Equivalent) Stains the Nucleus Overlay Doxorubicin (590/50) Differential Interference Contrast Figure 11: The free doxorubicin polymer appears to stain the mitochondria as well, but also exhibits diffuse cellular staining. In addition, strong doxorubicin fluorescence is observed in the nucleus; notably, the doxorubicin polymer did not display nuclear fluorescence (Figure 10).

15 Figure 12: The Doxorubicin Maleimide Polymer (pseudocolored red) appears to be trafficked to the mitochondria and qualitatively colocalizes with MitoTracker Deep Red (pseudocolored blue) in hbmmscs. Note the high degree of overlap as shown by the purple pixels in the overlay.

16 Figure 13: The Doxorubicin Maleimide Polymer quantitatively colocalizes with MitoTracker Deep Red in hbmmscs, with a Pearson s correlation of 0.75 and a Mander s overlap of 0.76 (Right graph). A region of interest was drawn in order to reduce background (left figure). Note the high degree of colocalized red pixels as shown in the overlay.