Block Copolymer Assemblies for Delivering Drugs and Bioimaging Agents

Transcription

1 Block Copolymer Assemblies for Delivering Drugs and Bioimaging Agents J. S. Riffle, N. Pothayee, R. Zhang, N. Hu, S. Roy- Chaudrury & R. M. Davis Macromolecules and Interfaces Laboratory Virginia Tech N. Pothayee & A. P. Koretsky, Laboratory for Functional and Molecular Imaging, NIH In honor of Jim McGrath San Michele

2 Objectives Design complexes that can carry and release cancer therapeutics with simultaneous MRI positive imaging Potentially track biodistribution of drug complexes in vivo

3 Outline for today Poly(ammonium bisphosphonate copolymers and manganese graft ionomer complexes (MaGICs) Strong binding of polymer to manganese little to no release in PBS and no displacement by calcium ions High relaxivities and potentially good image contrast Stable colloidal stability Early toxicity tests are promising Co-encapsulation of anticancer drugs and manganese ions into MaGICs for potential simultaneous imaging and delivery Good uptake of cancer drugs with release rates dependent on the drug structure Good anticancer efficacies against breast and brain (glioma) cancer cell lines

4 Magnetic Resonance Imaging (MRI) as a Tool for High Resolution In Vivo Imaging Contrast Agent-assisted MRI Loss signal/dark image = negative contrast Facilitates decay of signal due to T 2 -relaxation liver liver Pre-contrast Post-contrast Gain signal/bright image = posititive contrast Facilitates recovery of signal due to T 1 - relaxation liver liver 4 Pre-contrast Post-contrast

5 Manganese-based Contrast Agents as Alternatives to Gadolinium Mn spin number 5/2 Labile water exchange Natural cellular components Approved Mn contrast agents Mn-DPDP (dipyridoxyl diphosphate) Mn-DPDP (Teslascan) Although no relation between Mn and nephrogenic systemic fibrosis has been found so far, the free form of Mn 2+ is known to pose some neurotoxicity. However, this issue can be solved by chelation of free Mn 2+ with a chelating agent or polymeric nanomaterial to form a stable complex.

6 Comparison of Carboxylates vs Phosphonates Carboxyl 52:48 wt:wt PAA:PEO Propyl 60:40 wt:wt PABP:PEO Hexyl 59:41 wt:wt PABP:PEO Molecular weight of PEO = 5000 g/mole

7 β-aminobisphosphonate monomers

8 Polyaminobisphosphonate-g-PEO M n = 5K

9 Synthesis of MaGICs Mn 2+ DI water ph 7.4 Stirred for 24 h Dialyzed for 2 days Poly(ammonium bisphosphonate)-g-peo MaGICs Complex Moles of P/Mn or C/Mn Intensity average diameter (nm) Zeta Potential (mv) Carboxyl MaGICs Propyl MaGICs Hexyl MaGICs Carboxyl MaGICs Propyl MaGICs Hexyl MaGICs

10 Dispersions of Phosphonate MaGICs are Stable in PBS, ph 7.4 at RT but Carboxyl MAGICs are Not Intensity average diameter (nm) Propyl MaGICs 2.0 Propyl MaGICs 3.3 Hexyl MaGICs 2.0 Hexyl MaGICs 3.3 Carboxyl MaGICs 2.0 Carboxyl MaGICs Time (h) The MaGICs had excellent colloidal stability in phosphate buffered saline (PBS) for up to 24 h. This suggests that they will be sufficiently stable under physiological conditions to be suitable contrast agent.

11 % Accumulated release Mn in the Phosphonate MaGICs does not Release in PBS (ph 7.4 at 37 C) but it is Unstable in Carboxyl MAGICs % Propyl-MaGICs 3.3 Hexyl-MaGICs Carboxyl MaGICs 3.3 (control) MnCl % Time (h) 7 % 0 % At ph 7.4, Propyl and Hexyl MaGICs significantly slow down the release of Mn reaching ~ 0-7% in 24 h compared to Carboxyl MaGICs that release ~45 % of Mn within 24 h and free Mn that fully diffused through the dialysis membrane within 9 hours

12 NMR Relaxivities of MaGICs Measured at 1.4 Tesla, 37 C Complex Intensity ave Moles of P/Mn or C/Mn r 1 r 2 /r diameter 1 (nm) MnCl Carboxyl MaGICs Propyl MaGICs Hexyl MaGICs Carboxyl MaGICs Propyl MaGICs Hexyl MaGICs Mn-DPDP

13 T 1 - & T 2 -weighted Phantom Images (7 T, 25 C) T 1 -weighted T 2 -weighted μm metal ion GdDTPA MnDPDP Propyl MaGICS 2.0 Propyl MaGICs 3.3 Hexyl MaGICS 2.0 Hexyl MaGICS 3.3

14 In vivo MR imaging of MaGICs 15 μmol/kg MaGICs-CBPt 7.7 i.v. Pre-injection 40 min L S K 15 μmol/kg GdDTPA i.v. Bruker Pharmascan 7T, 16 cm bore Pre-injection 40 min GdDTPA MRI in C57BL6 mice at 7 T revealed excellent contrast signal enhancement (30-40%) exerted by the MaGICs at a dose fold lower than clinical dosage of Gd-based agents L = liver, S = spleen, K = kidney

15 R Ca 2+ does not displace the Mn 2+ and doesn t affect the relaxivities of MaGICs Measured at 1.4 Tesla, 37 C r 2 r mm (Mn) MnCl 2 Hexyl MaGICs (3.3) Hexyl MaGICs (3.3) mm Ca Since Ca 2+ is the most plentiful mineral found in the human body, stability of the MaGICs against Ca 2+ displacement is important! R mm (Mn)

16 Encapsulation of anticancer drugs into MaGICs PB, ph 7.4 Doxorubicin (DOX) 3 h MaGICs-DOX 9:1 water:dmso Cisplatin (CPt) 12 h MaGICs-CPt Hexyl MaGICs 3.3 PB, ph h Carboplatin (CBPt) MaGICs-CPt

17 Platinum drug-loaded MaGICs MaGICs-Cisplatin MaGICs-Carboplatin H 3 N H 3 N Pt Cl Cl H 3 N H 3 N Pt O O O O fast ligand exchange reaction formed two coordination bonds No covalent reaction strong interactions: electrostatic interactions + chelation

18 % Accumulated release % Accumulated release Release of Carboplatin in PBS is Significantly Faster than Cisplatin, ph 7.4 at 37 C 120 Carboplatin 120 Cisplatin % Free CBPt MaGICs-CBPt 13.0 MaGICs-CBPt % Free CPt MaGICs-CPt 5.0 MaGICs-CPt MaGICs-CBPt MaGICs-CPt Time (h) 34 % 22 % 11 % Time (h) 13 % 3 % 0 % The release rate of carboplatin from MaGICs was found to be faster than that of cisplatin but slower than doxorubicin

19 % Prolifera on MaGICs-Carboplatin enhance anti-proliferative effects relative to free carboplatin by 30-fold against breast cancer cells MaGICs-Cisplatin MaGICs-CPt = 80 μm Free-CPt = 20 μm MaGICs-CPt Pt concentra on (μm) CPt % Prolifera on Pt concentra on (μm) Cytotoxic effect against MCF-7 breast cancer cell using MTT assay MaGICs-Carboplatin MaGICs-CBPt = 3 μm Free-CBPt = 90 μm MaGICs-CBPt 13.0 CBPt

20 % Proliferation In Vitro Screening on U251 Glioblastoma Cells Showed Enhanced Anti-proliferative Activities Pt (um) cisplatin carboplatin MaGICs-CBPt 4.7 MaGICs-CBPt 7.7 MaGICs-CBPt 13 IC 50 (μm) cisplatin 7.0 carboplatin 50 MaGICs-CBPt MaGICs-CBPt MaGICs-CBPt estimated from Graphpad software *Measured by MTT assay after continuous exposure for 48 h

21 % Proliferation Pt pg/cell Increase in anti-proliferative activity is not due to enhanced intracellular uptake of Pt Intracellular Pt accumulation after 4 h incubation MaGICs-CBPt Carboplatin Pt (um) 4 h exposure to complexes and free drug followed by 44 h of incubation 0 Carboplatin MaGICs-CBPt 13.0 * Measured by ICP of total 1,000,000 cell lysates

22 % Proliferation The MaGICs-platinum complexes also show greater activities in cisplatin-resistant U251 (CR-U251) Pt (um) MaGICs-CBPt 12 MaGICs-CBPt 5.0 cisplatin CR-U251 were derived by culturing U251 in a low concentration of cisplatin for 5 months CR-U251 acquires 25-fold increase of cisplatin resistance. IC 50 of cisplatin in U251 is 6-7 um but in CR-U251 IC 50 of cisplatin is ~ um. MaGICs-CbPt complexes show an ~8-fold improved antiproliferative activity compared to cisplatin

23 Summary Complexation of Mn 2+ with poly(ammonium bisphosphonate)-g-peo significantly increases r 1 and decreases the r 2 /r 1, thus improving the contrast MaGICs essentially eliminate release of the Mn compared to Carboxylate complexes Physiological concentration of Ca 2+ does not displace the Mn from the complexes Carboplatin-loaded MaGICs enhanced anti-proliferative effects of - MCF-7 breast cancer cells ~30-fold - U251 glioblastoma cells - Cisplatin-resistant glioblastoma cells

24 Acknowledgements Nipon Pothayee Nikorn Pothayee Nan Hu Rui Zhang Sharavanan Balasubramaniam 24