Aptamer Directed Synergistic Drug Delivery

Transcription

1 Aptamer Directed Synergistic Drug Delivery Valerie Lensch Chemistry Mentor Anusha Pusuluri Samir Mitragotri Department of Chemical Engineering Tom Soh Department of Mechanical Engineering 1

2 Targeted Drug Delivery Minimizes Side Effects of Chemotherapeutic Drugs Drug 2

3 Targeted Drug Delivery Minimizes Side Effects of Chemotherapeutic Drugs Targeted Drug 2

4 Using Unique Characteristics of Breast Cancer Cells as Targets HR=Hormone Receptor HER2= Human Epidermal Growth Factor Receptor HR 3

5 Using Unique Characteristics of Breast Cancer Cells as Targets HR=Hormone Receptor HER2= Human Epidermal Growth Factor Receptor Overexpressed Nucleolin Receptors 3

6 Oligonucleotide Aptamer AS1411 Targets Biomarkers on Cancer Cells Peptide-Drug-Aptamer 4

7 Oligonucleotide Aptamer AS1411 Targets Biomarkers on Cancer Cells Peptide-Drug-Aptamer ssdna 4

8 Oligonucleotide Aptamer AS1411 Targets Biomarkers on Cancer Cells Overexpressed Nucleolin Receptors Peptide-Drug-Aptamer 4

9 Aptamer Constructs are Taken Up Through Nucleolin Receptor-Mediated Endocytosis Aptamer Construct Nucleolin Receptors Nucleus 5

10 Aptamer Constructs are Taken Up Through Nucleolin Receptor-Mediated Endocytosis Aptamer Construct Nucleolin Receptors Endosome Nucleus 5

11 Aptamer Constructs are Taken Up Through Nucleolin Receptor-Mediated Endocytosis Aptamer Construct Nucleolin Receptors Endosome Nucleus ph Drop 5

12 Aptamer Constructs are Taken Up Through Nucleolin Receptor-Mediated Endocytosis Aptamer Construct Nucleolin Receptors Endosome Nucleus ph Drop Drug 5

13 Combination Treatment Improves Efficacy of Therapy as Compared to Single Drug Treatment Cameron, et al. Breast cancer research and treatment (2008). 6

14 Drug Combinations Can Enhance or Undermine Drug Effect CI = 1 % Inhibition Drug A Drug B Additive Antagonistic Synergistic 7

15 Drug Combinations Can Enhance or Undermine Drug Effect CI = 1 % Inhibition CI > 1 Drug A Drug B Additive Antagonistic Synergistic 7

16 Drug Combinations Can Enhance or Undermine Drug Effect CI < 1 CI = 1 % Inhibition CI > 1 Drug A Drug B Additive Antagonistic Synergistic 7

17 Camptothecin and Doxorubicin Inhibit Topoisomerase I and II, Leading to Apoptosis Parent DNA Topoisomerase Daughter DNA Strand 8

18 Camptothecin and Doxorubicin Inhibit Topoisomerase I and II, Leading to Apoptosis Parent DNA Drug Topoisomerase 8

19 Drug Ratios Cause Synergism and Antagonism Combination Index R (CPT:DOX) 9 Camacho, K. M., et al. Journal of Controlled Release 210 (2015)

20 Drug Ratios Cause Synergism and Antagonism Combination Index R (CPT:DOX) 9 Camacho, K. M., et al. Journal of Controlled Release 210 (2015)

21 Drug Ratios Cause Synergism and Antagonism Combination Index R (CPT:DOX) 9 Camacho, K. M., et al. Journal of Controlled Release 210 (2015)

22 Determining the Synergistic Ratio of Doxorubicin and Camptothecin Free Drug Peptide-Drug Peptide-Drug-Aptamer 10

23 Using MTT Absorbance to Measure Cytotoxicity Seed Cells in 96 Well Plate & Incubate Overnight 11

24 Using MTT Absorbance to Measure Cytotoxicity Seed Cells in 96 Well Plate & Incubate Overnight Add Drug Combinations with Different Concentrations & Incubate for 72 Hours 11

25 Using MTT Absorbance to Measure Cytotoxicity Seed Cells in 96 Well Plate & Incubate Overnight Add Drug Combinations with Different Concentrations & Incubate for 72 Hours Remove Drug & Add Tetrazolium Dye MTT Solution until Purple Crystals Form 11

26 Using MTT Absorbance to Measure Cytotoxicity Seed Cells in 96 Well Plate & Incubate Overnight Add Drug Combinations with Different Concentrations & Incubate for 72 Hours Remove Drug & Add Tetrazolium Dye MTT Solution until Purple Crystals Form Dissolve MTT Crystals in DMSO & Measure Absorbance 11

27 MTT Plate Gradient Correlates Concentration and Cytotoxicity Decreasing Drug Concentration Control 12

28 CI s for Free Drug in MDA MB Combination Index R (CPT:DOX) 13

29 Synthesis of Peptide-Drug Constructs 14

30 Using Mass Spectrometry and TLC to Verify Pep-DOX Reaction C:\Users\massspec\Documents\MIT_ANP\MIT070716\MIT070716LB\0_F6\1\1SRef Comment 1 Pusuluri/Mitragotri/PD(Mal)_Impure_BOP-Cl Comment 2 ACHCA/TA30/Suppression_1000Da Intens. [a.u.] PEP-DOX PEP-DOX PEP-DOX DOX PEP PD m/z printed: 7/7/ :38:21 15

31 Intens. [a.u.] Using Mass Spectrometry and TLC to Verify Pep-CPT Reaction C:\Users\massspec\Documents\MIT_ANP\MIT062916\MIT062916LJ\0_D6\1\1SRef Comment 1 Pusuluri/Mitragotri/PD(DBCO)_PC(Mal)_ 20% MeCN Wash 2 Comment 2 ACHCA/TA30/Suppression_300 Da x PEP CPT PEP m/z C PC Bruker Daltonics flexanalysis printed: 7/5/2016 4:22:56 PM 16

32 Synthesis and Purification of Aptamer Constructs Using Click Chemistry Peptide-Drug 17

33 Synthesis and Purification of Aptamer Constructs Using Click Chemistry Peptide-Drug Purified Peptide-Drug C-18 (HPLC) Column 17

34 CI s for Peptide-Drug in MDA MB 231 Combination Index R (CPT:DOX) 18

35 Synthesis and Purification of Aptamer Constructs Using Click Chemistry Peptide-Drug Purified Peptide-Drug C-18 (HPLC) Column Peptide-Drug- Aptamer 19

36 Synthesis and Purification of Aptamer Constructs Using Click Chemistry Peptide-Drug Purified Peptide-Drug Purified Peptide-Drug- Aptamer C-18 (HPLC) Column Peptide-Drug- Aptamer Sodium Ethanol Precipitation G-10 (Gravity) Column 19

37 Using Gel Electrophoresis to Verify Aptamer-Peptide-Drug Conjugation 20

38 Confocal Microscopy Z Stack of Aptamer Construct Uptake by MCF 10A 21

39 Confocal Microscopy Z Stack of Aptamer Construct Uptake by MDA MB

40 Future Work Determining the Synergistic Ratio of Doxorubicin to Camptothecin for the Peptide-Drug-Aptamer Comparing Cytotoxicity of Peptide-Drug-Aptamer with Nonspecific Peptide-Drug-Aptamer 23

41 Acknowledgements Anusha Pusuluri Samir Mitragotri Chemical Engineering Tom Soh Mechanical Engineering Mellichamp Chair Funds Mitragotri Group Members Eureka Supermentors, Mentors, Peer Mentors 24

42 CI s for Free Drug in MCF 10A Combination Index R (CPT:DOX) 25

43 CI s for Peptide-Drug in MCF 10A Combination Index R (CPT:DOX) 26

44 Chou-Talalay Method CI = (D) 1 (D m ) 1 [f a /(1 f a )] 1/m1 + (D) 2 (D m ) 2 [f a /(1 f a )] 1/m2 27

45 Combination Treatment Improves Efficacy of Therapy as Compared to Single Drug Treatment Cameron, et al. Breast cancer research and treatment (2008). 28

46 Comment 1 Comment 2 Verification of Multiple Camptothecin Conjugation Pusuluri/Mitragotri/PD(Mal)_Impure_BOP-Cl C:\Users\massspec\Documents\MIT_ANP\MIT070716\MIT070716LB\0_F6\1\1SRef ACHCA/TA30/Suppression_1000Da Intens. [a.u.] PEP-DOX PEP-DOX PEP-DOX m/z printed: 7/7/ :38:21 29