Poly(L-lysine isolphthalamide) (PLP) polymers with hydrophobic pendant chains

Abstract

The present disclosure relates to the provision of novel biodegradable amphiphilic peptides and peptide analogues based derivatives comprising hydrophobic chains and their use in the permeabilization of mammalian cells and delivery of agents, for example therapeutic agents, imaging agents and cell preservation agents.

Claims

1. A poly(lysine isophthalamide) derivative comprising general formula (I): ##STR00004## wherein R comprises NR.sup.1R.sup.2 or OH wherein at least one of R are NR.sup.1R.sup.2; R.sup.1 and R.sup.2 each independently comprises: H; C.sub.6-30 alkyl, C.sub.6-30 alkenyl or C.sub.6-30 alkynyl, C.sub.6-10 aryl or C.sub.5-10 heteroaryl; wherein Alkyl, alkenyl and alkynyl groups R.sup.1 and R.sup.2 are optionally substituted with one or more substituents selected from halo, cyano, nitro, diazonium, —OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4, NR.sup.3R.sup.4, ═NR.sup.3, ═O, C(O)OR.sup.3, OR.sup.3, SR.sup.3, C(O)SR.sup.3, C(O)NR.sup.3R.sup.4, azide, C.sub.3-7 cycloalkyl, C.sub.3-10 heterocyclyl, C.sub.6-14 aryl or C.sub.4-14 heteroaryl; wherein cycloalkyl, heterocyclyl, aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C.sub.1-10 alkyl, C.sub.1-10 haloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4, aryl substituted with R.sup.3 and heteroaryl substituted with R.sup.3 and, where chemically appropriate, ═O; and wherein each of R.sup.3 and R.sup.4 is independently H or C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.6-10 aryl; aryl and heteroaryl groups R.sup.1 and R.sup.2 are optionally substituted with one or more substituents selected from C.sub.1-16 alkyl, halo, cyano, nitro, diazonium, —OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, C(O)SR.sup.3, C(O)NR.sup.3R.sup.4, azide, C.sub.6-14 aryl, C.sub.4-14 heteroaryl or S—CH.sub.2C(O)NR.sup.5R.sup.6; wherein alkyl groups are optionally substituted with one or more substituents selected from halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4; wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C.sub.1-10 alkyl, C.sub.1-10 haloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4; wherein R.sup.3 and R.sup.4 are as defined above and R.sup.5 and R.sup.6 are each independently H, C.sub.1-6 alkyl optionally substituted with OR.sup.3 or halo or C.sub.6-14 aryl optionally substituted with C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl) or O—C.sub.6-14 aryl; or R.sup.1 and R.sup.2 together with the nitrogen atom to which they are attached form a 5-12-membered heterocyclic ring optionally containing one or more additional heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected from C.sub.1-16 alkyl, C.sub.1-16 haloalkyl, halo, cyano, nitro, diazonium, ═O, —OP(O)OR.sup.3R.sup.4, —PR.sup.3R.sup.4, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, C(O)SR.sup.3, C(O)NR.sup.3R.sup.4, azide, C.sub.6-14 aryl or C.sub.4-14 heteroaryl; wherein alkyl and haloalkyl groups are optionally substituted with one or more substituents selected from halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C.sub.1-10 alkyl, C.sub.1-10 haloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4; wherein R.sup.3 and R.sup.4 are as defined above; and n≥4.

2. A poly(lysine isophthalamide) derivative according to claim 1 wherein R.sup.1 and R.sup.2 each independently comprises: H; C.sub.6-30 alkyl, C.sub.6-30 alkenyl or C.sub.6-30 alkynyl group optionally substituted with one or more substituents selected from halo, cyano, nitro, diazonium, —OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, C(O)SR.sup.3, C(O)NR.sup.3R.sup.4, azide, C.sub.6-14 aryl or C.sub.4-14 heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C.sub.1-10 alkyl, C.sub.1-10 haloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4; and wherein each of R.sup.3 and R.sup.4 is independently H or C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.6-10 aryl; C.sub.6-10 aryl optionally substituted with one or more substituents selected from C.sub.1-16 alkyl, C.sub.1-16 haloalkyl, halo, cyano, nitro, diazonium, —OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, C(O)SR.sup.3, C(O)NR.sup.3R.sup.4, azide, C.sub.6-14 aryl or C.sub.4-14 heteroaryl, wherein alkyl and haloalkyl groups are optionally substituted with one or more substituents selected from halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4; wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C.sub.1-10 alkyl, C.sub.1-10 haloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4; wherein R.sup.3 and R.sup.4 are as defined above; or R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached to form a 5-12-membered heterocyclic ring optionally containing one or more additional heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected from C.sub.1-16 alkyl, C.sub.1-16 haloalkyl, halo, cyano, nitro, diazonium, —OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, C(O)SR.sup.3, C(O)NR.sup.3R.sup.4, azide, C.sub.6-14 aryl or C.sub.4-14 heteroaryl; wherein alkyl and haloalkyl groups are optionally substituted with one or more substituents selected from halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C.sub.1-10 alkyl, C.sub.1-10 haloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, azide, OP(O)OR.sup.3OR.sup.4, —PR.sup.3R.sup.4; wherein R.sup.3 and R.sup.4 are as defined above; and n≥4.

3. A poly(lysine isophthalamide) derivative according to claim 1 wherein R.sup.1 is as defined in claim 1 and R.sup.2 is C.sub.6-30 alkyl, C.sub.6-30 alkenyl or C.sub.6-30 alkynyl, C.sub.6-10 aryl or C.sub.5-10 heteroaryl, any of which is optionally substituted as defined in claim 1.

4. A poly(lysine isophthalamide) derivative according to claim 3, wherein R.sup.1 is H, C.sub.6-30 alkyl, C.sub.6-30 alkenyl or C.sub.6-30 alkynyl, any of which may optionally be substituted as defined in claim 1 and R.sup.2 is C.sub.6-30 alkyl, C.sub.6-30 alkenyl or C.sub.6-30 alkynyl, any of which may optionally be substituted as defined in claim 1.

5. A poly(lysine isophthalamide) derivative according to claim 4 wherein R.sup.1 is H or C.sub.6-30 alkyl, C.sub.6-30 alkenyl or C.sub.6-30 alkynyl, any of which is unsubstituted or is substituted with F, Cl, OH, SH, methoxy or ethoxy; and R.sup.2 is C.sub.6-30 alkyl, C.sub.6-30 alkenyl or C.sub.6-30 alkynyl, any of which is unsubstituted or is substituted with F, Cl, OH, SH, methoxy or ethoxy.

6. A poly(lysine isophthalamide) derivative according to claim 5 wherein R.sup.1 is H or unsubstituted C.sub.6-30 alkyl, unsubstituted C.sub.6-30 alkenyl or unsubstituted C.sub.6-30 alkynyl; and R.sup.2 is unsubstituted C.sub.6-30 alkyl, unsubstituted C.sub.6-30 alkenyl or unsubstituted C.sub.6-30 alkynyl.

7. A poly(lysine isophthalamide) derivative according to claim 5, wherein R.sup.1 is H and R.sup.2 is unsubstituted C.sub.7-18 alkyl.

8. A poly(lysine isophthalamide) derivative according to claim 7, wherein each of R.sup.1 and R.sup.2 is independently unsubstituted C.sub.7-18 alkyl.

9. The poly(lysine isophthalamide) derivative according to claim 7 wherein R.sup.2 is n-heptyl, n-decyl, n-tetradecyl or n-octadecyl.

10. The poly(lysine isophthalamide) derivative according to claim 9 wherein said R.sup.2 is n-decyl.

11. The poly(lysine isophthalamide) derivative according to claim 1 wherein between 0.1-99% of the moieties R are NR.sup.1R.sup.2.

12. The poly(lysine isophthalamide) derivative according to claim 11 wherein 3-18% of the moieties R are NR.sup.1R.sup.2.

13. The poly(lysine isophthalamide) derivative according to claim 2 wherein each of R.sup.1 and R.sup.2 is C.sub.7-18 alkyl, C.sub.7-18 alkenyl or C.sub.7-18 alkynyl, any of which may optionally be substituted as defined in claim 2.

14. The poly(lysine isophthalamide) derivative according to claim 13 wherein each of R.sup.1 and R.sup.2 is C.sub.7-alkyl, C.sub.8-alkyl, C.sub.10 alkyl, C.sub.14 alkyl or C.sub.18 alkyl, any of which may be optionally be substituted according to claim 2.

15. The poly(lysine isophthalamide) derivative according to claim 1 wherein each of R.sup.1 and R.sup.2 is optionally substituted with one or more substituents selected from halo, cyano, nitro, azo, diazonium, phosphate, phosphate ester, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, C(O)SR.sup.3, C(O)NR.sup.3R.sup.4, azide, C.sub.6-14 aryl or C.sub.4-14 heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C.sub.1-10 alkyl, C.sub.1-10 haloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, halo, cyano or nitro, NR.sup.3R.sup.4, C(O)OR.sup.3, OR.sup.3, SR.sup.3, RN.sub.3, phosphate, phosphate ester and wherein each of R.sup.3 and R.sup.4 is independently H or C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10alkynyl.

16. The poly(lysine isophthalamide) derivative according to claim 2 wherein each of R.sup.1 and R.sup.2 is optionally substituted with one or more substituents selected from halo, cyano, nitro, NR.sup.3R.sup.4, C(O)OR.sub.3OR.sup.3SR.sup.3, C.sub.6-10 aryl or heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, halo, cyano or nitro; and wherein each of R.sup.3 and R.sup.4 is independently H or C.sub.1-6 alkyl.

17. The poly(lysine isophthalamide) derivative according to claim 1 wherein said peptide is associated, either directly or indirectly with an agent for intracellular delivery to a cell.

18. The poly(lysine isophthalamide) derivative according to claim 17 wherein said agent is covalently or non-covalently associated with said poly(lysine isophthalamide) derivative.

19. The poly(lysine isophthalamide) derivative according to claim 18 wherein said agent is a therapeutic agent.

20. The poly(lysine isophthalamide) derivative according to claim 18 wherein said agent is an imaging agent.

21. The poly(lysine isophthalamide) derivative according to claim 18 wherein said agent is a cell preservation agent.

22. A process for the preparation of poly(lysine isophthalamide) derivative according to claim 1 comprising the steps i) polymerization of aqueous lysine methyl ester.2HCl with an equivalent amount of isophthaloyl chloride in acetone and subsequent hydrolysis in DMSO with ethanolic sodium hydroxide, and ii) conjugation of R, wherein R comprises NR.sup.1R.sup.2 and is defined as above onto the polymer backbone via dicyclohexylcarboiimide/dimethylaminopyridine (DCC/DMAP) coupling; or conjugation of R is via 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) coupling.

23. A composition comprising a poly(lysine isophthalamide) derivative according to claim 1.

24. The composition according to claim 23 wherein the agent is a therapeutic agent and the composition is a pharmaceutical composition including a pharmaceutically acceptable carrier.

25. The composition according to claim 23 wherein said composition comprises mammalian cells or a collection of mammalian cells.

26. A composition according to claim 23 for use in the delivery of at least one agent to a mammalian cell, cellular aggregate, tissue or organ.

27. An in vitro or ex vivo method to deliver an agent to a cell comprising: i) contacting cells or a cellular aggregate, tissue or organ comprising cells with an effective amount of a composition according to claim 23; and ii) incubating said cell, cellular aggregate, tissue or organ to allow permeabilization of said mammalian cells or cellular aggregate, tissue or organ comprising cells thereby delivering said agent.

28. The method according to claim 27 wherein said cell is a mammalian cell.

29. An in vitro or ex vivo method for the preservation of a mammalian cell, cellular aggregate, tissue or organ comprising the steps: i) providing a preparation comprising a mammalian cell preparation, mammalian cellular aggregate, tissue or organ and a composition according to claim 23; ii) incubating said preparation to permeabilize the mammalian cell membranes of said mammalian cell, cellular aggregate, tissue or organ; and iii) contacting said permeabilized cell, cellular aggregate, tissue or organ with one or more preservation agents.

30. The method according to claim 29 wherein said preservation agent is a sugar.

Description

(1) An embodiment of the invention will now be described by example only and with reference to the following figures:

(2) FIGS. 1(A)-(B): FIG. 1(A) 1H-NMR spectra of PLP grafted with NDA in acid form in de-DMSO at room temperature. FIG. 1(B) FTIR spectra of PLP grafted with NDA in acid form. In addition to NDA (C10), other pendant chains including HDA (C7), TDA (C14) and ODA (C18) have also been conjugated to the pseudo-peptide backbone;

(3) FIGS. 2(A)-(C): FIG. 2(A) pH dependent transmittance of the aqueous solutions of PLP (.square-solid.), PLP-NDA 3% (.circle-solid.), PLP-NDA 10% (.box-tangle-solidup.) and PLP-NDA 18% (.Math.) at 1.0 mg mL.sup.−1 in 100 mM buffers. FIG. 2(B) pH dependent change of I.sub.338/I.sub.333 in the excitation spectra of pyrene dissolved in aqueous solutions of PLP (.square-solid.), PLP-NDA 3% (.circle-solid.), PLP-NDA 10% (.box-tangle-solidup.), PLP-NDA 18% (.Math.) at 0.5 mg mL.sup.−1. FIG. 2(C) Concentration dependent change of I.sub.338/I.sub.333 in the excitation spectra of pyrene dissolved in aqueous solutions of PLP (.square-solid.), PLP-NDA 3% (.circle-solid.), PLP-NDA 10% (.box-tangle-solidup.), PLP-NDA 18% (.Math.) at pH 7.4;

(4) FIGS. 3(A)-(B): Particle size distributions of FIG. 3(A) PLP and FIG. 3(B) PLP-NDA 18% at the concentration of 0.5 mg mL.sup.−1 at pH 7.4 (solid) and pH 5.5 (dash);

(5) FIGS. 4(A)-(C): Relative haemolysis of red blood cells with the present of PLP and its derivatives. FIG. 4(A) pH dependent haemolysis of RBC incubated with PLP (.square-solid.), PLP-NDA 3% (.circle-solid.), PLP-NDA 10% (.box-tangle-solidup.), PLP-NDA 18% (.Math.) at 0.5 mg mL.sup.−1 for 1 h. FIG. 4(B) Concentration dependent haemolysis of RBC incubated with PLP-NDA 18% at pH 7.4 (open column) and pH 5.5 (closed column). FIG. 4(C) Time dependent haemolysis of RBC incubated with PLP (.square-solid.), PLP-NDA 3% (.circle-solid.), PLP-NDA 10% (.box-tangle-solidup.), PLP-NDA 18% (.Math.) at 0.5 mg mL.sup.−1 at pH 5.5;

(6) FIGS. 5(A)-(D): In-vitro cytotoxicity. Viability of FIG. 5(A) HeLa cells, FIG. 5(B) CHO cells and FIG. 5(C) A549 cells incubated with PL-NDA 18% at various concentrations for 4 h (blank), 12 h (grey), 24 h (black) and 48 h (stripped). FIG. 5(D) In-vitro cytotoxicity of PLP (blank) and PLP-NDA 18% (grey) against HeLa, CHO and A549 cells at polymer concentration of 0.5 mg mL.sup.−1 for 24 h;

(7) FIGS. 6(A)-(C): Confocal microscopy images of FIG. 6(A) Hela cells. FIG. 6(B) CHO cells and FIG. 6(C) A549 cells showing the subcellular distribution of calcein fluorescence. The cells were treated with 2.0 mg mL.sup.−1 calcein alone, both 2.0 mg mL.sup.−1 calcein and 0.5 mg mL.sup.−1 PLP, or both 2.0 mg mL.sup.−1 calcein and 0.5 mg mL.sup.−1 PLP-NDA 18% respectively. Images of HeLa and CHO cells were acquired after 1 h of uptake and further incubated for 3 h. For A549 cells, the uptake was 2 h and the further incubation was 2 h. Scale bar: 10 μm;

(8) FIGS. 7(A)-(C): PLP-NDA 18% mediated delivery of FITC-dextran with different molecular weights into HeLa cells. FIG. 7(A) HeLa cells incubated with 0.5 mg mL.sup.−1 PLP-NDA 18% and FITC-dextran at pH 6.5 for 30 min. FIG. 7(B) HeLa cells incubated with FITC-dextran only at pH 6.5 for 30 min. FIG. 7(C) HeLa cells incubated with 0.5 mg mL.sup.−1 PLP-NDA 18% and FITC-dextran at pH 7.4 for 30 min. Scale bar: 20 μm;

(9) FIG. 8: Polymer concentration-dependent intracellular delivery. HeLa cells were incubated with PLP-NDA 18% at various concentrations and 200 μM FITC-dextran (4 kDa) at pH 6.5 for 30 min. Scale bar: 20 μm.

(10) FIGS. 9(A)-(B): FIG. 9(A) Confocal microscopy images and FIG. 9(B) relative mean fluorescence intensity (MFI) of the polymer-mediated delivery analyzed by flow cytometry. HeLa cells were incubated with 200 μM FITC-dextran (4 kDa) in the absence (control) or in the presence of 0.5 mg mL.sup.−1 comb-like polymers containing alkyl chains with different lengths at pH 6.5 for 30 min. Scale bar: 20 μm. Mean±S.D. (n=3).

(11) FIG. 10. Confocal microscopy images of pH-dependent polymer-mediated intracellular delivery. HeLa cells were co-incubated with 0.5 mg mL.sup.−1 PLP-NDA 18% and 200 μM FITC-dextran (4 kDa) at various extracellular pHs for 30 min. Scale bar: 20 μm.

(12) FIGS. 11(A)-(B). FIG. 11(A) Relative MFI and FIG. 11(B) representative histogram plots of the polymer-mediated delivery of 200 μM FITC-dextran (4 kDa) at different extracellular pHs analyzed by flow cytometry. HeLa cells were incubated in the absence (open columns) or in the presence of PLP-NDA 18% at 0.5 mg mL.sup.−1 (dose columns) at different extracellular pHs for 30 min. Mean±S.D. (n=3).

(13) FIG. 12. Confocal microscopy images of time-dependent polymer-mediated intracellular delivery. HeLa cells were co-incubated with 0.5 mg mL.sup.−1 PLP-NDA 18% and 200 μM FITC-dextran (4 kDa) at pH 6.5 for various time periods. Scale bar: 20 μm.

(14) FIG. 13. Relative MFI of time-dependent polymer-mediated intracellular delivery analyzed by flow cytometry. HeLa cells were incubated with 200 μM FITC-dextran (4 kDa) in the absence (open columns) or in the presence of 0.5 mg mL.sup.−1 PLP-NDA 18% (close columns) at pH 6.5 for various time periods. Mean±S.D. (n=3).

(15) FIG. 14. Confocal microscopy images of polymer-mediated delivery of FITC-dextran (4 kDa) into difference cell types. All the cells were co-treated with 0.5 mg mL.sup.−1 PLP-NDA 18% and 200 μM FITC-dextran at pH 6.5. The treatment time was 30 min for HeLa, CHO and SU-DHL-8 cells, 180 min for A549 cells, and 60 min for MES-SA, MES-SA/DX5 and hMSCs. Scale bar: 20 μm.

(16) FIGS. 15(A)-(B). FIG. 15(A) Relative MFI and FIG. 15(B) representative histogram plots showing the polymer-mediated delivery of FITC-dextran into difference cell types. All the cells were treated with 0.5 mg mL.sup.−1 PLP-NDA 18% and 200 μM FITC-dextran (4 kDa) at pH 6.5. The treatment time was 30 min for HeLa, CHO and SU-DHL-8 cells, 180 min for A549 cells, and 50 min for MES-SA, MES-SA/DX5 and hMSC cells. Mean±S.D. (n=3).

(17) FIGS. 16(A)-(C). FIG. 16(A) Polymer concentration-dependent in vitro cytotoxicity. HeLa cells were treated with PLP-NDA 18% at various concentrations at pH 6.5 (close columns) and pH 7.4 (open columns) for 1 h. FIG. 16(B) Time-dependent in vitro cytotoxicity. HeLa cells were treated with PLP-NDA 18% at 0.5 mg mL.sup.−1 at pH 6.5 (close columns) and pH 7.4 (open columns) for various time periods. FIG. 16(C) In vitro cytotoxicity of PLP-NDA 18% toward a variety of cell lines. HeLa, A549, CHO, MES-SA, MES-SA/DX5, and hMSCs were treated with PLP-NDA 18% at 0.5 mg mL.sup.−1 at pH 6.5 (close columns) and pH 7.4 (open columns) for 3 h. Mean±S.D. (n=3).

(18) FIG. 17: Trehalose loading of erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) in 0.36 M trehalose solution and with addition of different concentrations of PLP-NDA 18%. [PLP-NDA 18%]=300, 450, 600 and 800 μg mL.sup.−1; incubation time=15 min, 30 min and 1 h; temperature=37° C. and pH=7.05. The intracellular trehalose concentration was calculated by the anthrone method. Data were derived from three replicates. Error bars represent standard deviations.

(19) FIG. 18: Haemolysis of erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) in 0.36 M trehalose solution and with addition of different concentrations of PLP-NDA 18%. [PLP-NDA 18%]=300, 450, 600 and 800 μg mL.sup.−1; incubation time=15 min, 30 min and 1 h; temperature=37° C. and pH=7.05. Supernatants were collected and the absorbance was measured by UV-VIS spectrophotometry at 541 nm. Data were derived from three replicates. Error bars represent standard deviations;

(20) FIG. 19: Trehalose loading of erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) in 0.36 M trehalose solution and with addition of different concentrations of PLP-NDA 18%. [PLP-NDA 18%]=600, 800, 1200 μg mL.sup.−1; incubation time=15 min, temperature=37° C. and pH=7.05, 6.8, 6.5, 6.1, 5.6. The intracellular trehalose concentration was calculated by the anthrone method. Data were derived from three replicates. Error bars represent standard deviations;

(21) FIG. 20: Haemolysis of erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) in 0.36 M trehalose solution and with addition of different concentrations of PLP-NDA 18%. [PLP-NDA 18%]=600, 800, 1200 μg mL-1; incubation time=15 min, temperature=37° C. and pH=7.05, 6.8, 6.5, 6.1, 5.6. Supernatants were collected and the absorbance was measured by UV-VIS spectrophotometry at 541 nm. Data were derived from three replicates. Error bars represent standard deviations;

(22) FIG. 21: Time dependent trehalose loading and haemolysis of erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) in 0.36 M trehalose solution and with addition of 800 μg mL.sup.−1 PLP-NDA 18%. Temperature=37° C. and pH=6.1. The intracellular trehalose concentration was calculated by the anthrone method. Supernatants were collected and the absorbance was measured by UV-VIS spectrophotometry at 541 nm. Data were derived from three replicates. Error bars represent standard deviations;

(23) FIG. 22: Temperature dependent trehalose loading and haemolysis of erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) in 0.36 M trehalose solution and with addition of 800 μg mL.sup.−1 PLP-NDA 18%. Incubation time=15 min and pH=6.1. The intracellular trehalose concentration was calculated by the anthrone method. Supernatants were collected and the absorbance was measured by UV-VIS spectrophotometry at 541 nm. Data were derived from three replicates. Error bars represent standard deviations:

(24) FIG. 23: Impact of extracellular trehalose concentration on trehalose loading and haemolysis of erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) in trehalose solution and with addition of 800 μg mL.sup.−1 PLP-NDA 18%. Incubation time=1 h, temperature=37° C. and pH=6.1. The intracellular trehalose concentration was calculated by the anthrone method. Supernatants were collected and the absorbance was measured by UV-VIS spectrophotometry at 541 nm. Data were derived from three replicates. Error bars represent standard deviations;

(25) FIG. 24: Impact of the length of hydrophobic pendant chains on trehalose loading. Erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) were treated in 0.36 M trehalose solution containing of 800 μg mL.sup.−1 PLP-HAD (7-carbon chain), PLP-NDA (10-carbon chain), PLP-TDA (14-carbon chain) and PLP-ODA (18-carbon chain). Incubation time=15 min; temperature=37° C. and pH=6.1. The intracellular trehalose concentration was calculated by the anthrone method. Data were derived from three replicates. Error bars represent standard deviations;

(26) FIG. 25: Impact of the length of hydrophobic pendant chains on haemolysis. Erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) were treated in 0.36 M trehalose solution containing of 800 μg mL.sup.−1 PLP-HAD (C7 chain), PLP-NDA (C10 chain), PLP-TDA (C14 chain) and PLP-ODA (C18 chain). Incubation time=15 min; temperature=37° C. and pH=6.1. Supernatants were collected and the absorbance was measured by UV-VIS spectrophotometry at 541 nm. Data were derived from three replicates. Error bars represent standard deviations;

(27) FIG. 26: Impact of the degree of grafting with the hydrophobic pendant chain NDA on trehalose loading and haemolysis. Erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per ml) were treated in 0.36 M trehalose solution containing of 800 μg mL.sup.−1 PLP-NDA at the degrees of grafting of 3%, 10% and 18%. Incubation time=15 min; temperature=37° C. and pH=6.1. The intracellular trehalose concentration was calculated by the anthrone method. Supernatants were collected and the absorbance was measured by UV-VIS spectrophotometry at 541 nm. Data were derived from three replicates. Error bars represent standard deviations;

(28) FIG. 27: Confocal microscopy images of polymer-mediated delivery into erythrocytes. Erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) were co-incubated with 0.36 M trehalose and 0.1 mM calcein in the absence or in the presence of 800 μg mL.sup.−1 PP50 (PLP grafted with L-phenylalanine) or PLP-NDA 18% at different pHs for 15 min, temperature=37° C. Scale bar: 2 μm.

(29) FIG. 28: Flow cytometry analysis of polymer-mediated delivery into erythrocytes. Erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) were incubated with 0.36 M trehalose and 0.1 mM calcein in the absence or in the presence of 800 μg mL.sup.−1 PP50 or PLP-NDA 18% at different pHs for 15 min, temperature=37° C. Mean±S.D. (n=3).

(30) FIGS. 29(A)-(B): Confocal microscopy images showing the membrane integrity after trehalose loading. FIG. 29(A) Erythrocytes incubated with 0.36 M trehalose in PBS buffer at pH 6.1 for 15 min, washed with pH 7.4 buffer twice and incubated with 1 μM calcein at pH 7.4. FIG. 29(B) Erythrocytes treated with 800 μg mL.sup.−1 PLP-NDA 18% and 0.36 M trehalose in PBS buffer at pH 6.1 for 15 min, washed with pH 7.4 buffer and incubated with 1 μM calcein at pH 7.4. Scale bar: 4 μm.

(31) FIG. 30: Topographic AFM micrographs of the erythrocyte membrane surface treated with different polymers. Erythrocytes (packed volume of 15%, 3.5×109 cells per mL) were incubated with 0.36 M trehalose solution in the absence or in the presence of 800 μg mL-1 PP50 or PLP-NDA 18% at pH 6.1 for 15 min at 37° C. The cells were immobilized on a polylysine coated microscope slide, crosslinked in glutaraldehyde (1%), washed three times with deionized water and then air dried. AFM was performed using the Asylum MFP-3D microscope in the tapping mode. Nanosensors PPP-NCHR tips (resonant frequency=app. 320 kHz nom. tip radius 7 nm, nom. Spring constant 42 N m-1) were used and tuned to a target tapping amplitude of 1-2 V.

(32) FIG. 31: Cryosurvival (%) of erythrocytes. Erythrocytes (packed volume of 15%, 3.5×10.sup.9 cells per mL) were suspended in 306 mOsm PBS buffer (.square-solid.), in 0.36 M extracellular trehalose solution at pH 7.05 (.circle-solid.), and in 0.36 M extracellular trehalose solution at pH=6.10 containing 800 μg mL.sup.−1 PLP-NDA 18% (.box-tangle-solidup.). Incubation time=15 min and temperature=37° C. After trehalose loading, erythrocytes were transferred into 2-mL cyrovial tubes followed by immersion into liquid nitrogen (−196° C.) for a certain period of time. The erythrocytes were then thawed in a 37° C. water bath for 15 min. Data were derived from three replicates. Error bars represent standard deviations.

(33) TABLE-US-00001 TABLE 1 pH values at the onset of precipitation (pH.sub.p), hydrophobic association (pH.sub.h), pH ranges for association and the critical association concentrations (CAC) of PLP and its derivatives. PLP-NDA PLP-NDA PLP-NDA PLP 3% 10% 18% pH.sub.p 4.5 4.5  4.5  5.0  pH.sub.h 4.8 ± 0.2 5.0 ± 0.2 6.0 ± 0.2 N/A pH range 3.5-4.8 4.0-5.0 3.5-6.0 N/A CAC (mg mL.sup.−1) N/A 0.342 0.282 0.031

(34) TABLE-US-00002 TABLE 2 The mean hydrodynamic diameters of PLP and its derivatives at concentration of 0.5 mg mL.sup.−1 at pH 7.4. PLP-NDA PLP-NDA PLP-NDA PLP 3% 10% 18% Population 1 10.3 ± 2.3   4.9 ± 1.2 22.7 ± 5.3  6.5 ± 2.0 mean size (nm) Population 2 384. ± 34.5 183.4 ± 12.6 151.3 ± 11.7 51.9 ± 1.6 mean size (nm)

(35) TABLE-US-00003 TABLE 3 The mean hydrodynamic diameters of PLP-NDA 18% with various concentrations at pH 7.4. 2 mg mL.sup.−1 1 mg mL.sup.−1 0.5 mg mL.sup.−1 0.1 mg mL.sup.−1 0.05 mg mL.sup.−1 Population 1 8.1 ± 1.6  6.3 ± 0.9  6.5 ± 2.0  6.3 ± 2.7  6.4 ± 3.6 mean size (nm) Population 2 N/A 28.3 ± 2.4 51.9 ± 1.6 102.1 ± 3.2 122.7 ± 12.6 mean size (nm)

(36) TABLE-US-00004 TABLE 5 Roughness Average (Ra) or Root Mean Square Roughness (RMS) of erythrocytes treated with different polymers. Erythrocytes (packed volume of 15%, 3.5 × 109 cells per mL) were incubated with 0.36M trehalose solution in the absence or presence of 800 μg mL.sup.−1 PP50 or PLP-NDA 18% at pH 6.1 for 15 min at 37° C. The cells were immobilized on a polylysine coated microscope slide, cross linked in glutaraldehyde (1%), washed three times with deionized water and then air dried, followed by the AFM measurement. Ra (nm) RMS (nm) Control 0.6101 0.8419 PP50 2.461 3.122 PLP-NDA 18% 17.16 21.35

(37) Materials and Methods

(38) Decylamine (NDA), heptylamine (HDA), tetradecylamine (TDA), octadecylamine (ODA), iso-phthaloyl chloride, fluorescein isothiocyanate-dextran (FITC-dextran, average Mw 4K, 10K, 70K, 150K and 2000K), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), MEM non-essential amino acid solution, Dulbecco's Phosphate Buffered Saline (D-PBS), penicillin and anthrone were purchased from Sigma Aldrich (Dorset, UK). Dimethyl sulfoxide (DMSO), pyrene, N,N-dimethylformamide (DMF), 4-dimethylaminopyridine (DMAP), AlamarBlue®, methanol (299.8%), sodium chloride, sodium phosphate dibasic heptahydrate, potassium chloride and potassium dihydrogen orthophosphate were obtained from Fisher Scientific (Loughborough, UK). lysine methyl ester dihydrochloride, N,N′-dicyclohexylcarbodiimide (DCC), triethylamine, ninhydrin and D-(+)-Trehalose dihydrate (≥99%) were purchased from Alfa Aesar (Heysham, UK). Anhydrous ethanol, acetone, hydrochloric acid, sodium hydroxide, chloroform, diethyl ether and sulphuric acid (≥95%) were obtained from VWR (Lutterworth, UK). Defibrinated sheep red blood cells (RBCs) were purchased from TCS Biosciences Ltd (Buckingham, UK), stored in a 4° C. refrigerator and used within one week once obtained.

(39) Poly(propylene glycol)-, polyethylene-, and polystyrene-based polymers are available from Sigma. Fatty acids are available from 3B Scientific Corporation. The following compounds were purchased from Sigma N-(2-Naphthyl)-1-naphthylamine (762660), c-(2-p-Tolyl-imidazo[1,2-a]pyridin-3-yl)-methylamine (CDS008330), 1,1-bis(4-chlorophenyl)-2-[(2-fluorobenzyl)amino]-1-ethanol (CDS018870), 4-Tetradecylaniline (233552), Bis[2-(di-tert-butylphosphino)ethyl]amine solution (739022), 3-(Fmoc-amino)benzonitrile (750352), h-cys(trt)-nh2 (CDS018559), 1,7-Dibenzyl-1,4,7,10-tetraazacyclododecane (CDS001040), 2-(3-oxo-decahydro-quinoxalin-2-yl)-N-(4-phenoxy-phenyl)-acetamide (CDS018799), pontacyl carmine 2b (CDS010534), 2-[(2-amino-4-methylphenyl)sulfanyl]-N-(2-methylphenyl)acetamide (CDS015863), 4-Nitrophenethylamine hydrochloride (184802), 3-(Ethoxydimethylsilyl)propylamine (588857), Decylamine (D2404), Octadecylamine (74750), Dihexylamine (131202), Dioctadecylamine (42358), 3-Butenylamine hydrochloride (597678), Oleylamine (O07805).

(40) Ethyl(prop-2-en-1-yl)amine (MolPort-000-005-271) but-3-yn-1-amine hydrochloride (MolPort-004-968-587), bis(but-2-yn-1-yl)amine (MolPort-001-991-305) and bis[(2Z)-3-chlorobut-2-en-1-yl]amine (MolPort-000-163-345) was purchased from MolPort.

(41) Methyl[7-(methylimino)hepta-1,3,5-trien-1-yl]amine (FCH4099593), 3-fluoro-2-methyloct-7-yn-1-amine (BBV-70832810), [4,4-dimethyl-2-(pent-4-yn-1-yl)cyclohexyl]methanamine (BBV-49722550), (8-aminoocta-1,3,5,7-tetrayn-1-yl)borane (FCH1957383), (1,2,3,6-tetrahydropyridin-4-yl)phosphonic acid was purchased from EnamineStore (EN300-298509) and (dec-1-yn-4-yl)(propyl)amine (CSC013776799) was purchased from Chemspace.

(42) Polymer Synthesis

(43) Synthesis of Poly(Lysine Isophthalamide) (PLP)

(44) PLP was synthesized using the single phase polymerization technique. In a typical procedure, lysine methyl ester.2HCl (0.15 mole) and potassium carbonate (0.6 mole) were dissolved in 750 mL of deionized water and stirred in an ice bath. To this was added rapidly 750 mL of a pre-cooled solution of anhydrous iso-phthaloyl chloride in dried acetone (0.2 M). The reaction was allowed to proceed until precipitation of poly(lysine methyl ester iso-phthalamide) (PLP methyl ester). The polymer was washed several times with deionized water, and dried overnight.

(45) 5 wt % NaOH solution in anhydrous ethanol (2.5 molar equivalents to PLP methyl ester) was added in several portions to a solution of PLP methyl ester in dry DMSO at the same volume (0.5 M). The hydrolysed product precipitated out in 2-3 minutes, and was collected by vacuum filtration and redissolved in deionized water. The crude polymer solution was dialysed in Visking tubing membrane (Medicell, MWCO 12-14 kDa) against deionized water to remove inorganic salts, residual organic solvents and low molecular weight oligomers. Solid impurities were removed by vacuum filtration. The clear solution was concentrated, adjusted to ˜pH 7.4 using a NaOH aqueous solution, and lyophilized to produce PLP in the sodium salt form. In order to prepare its neutral form, the dialysed polymer solution was acidified to pH˜3.0 with a dilute HCl solution. The precipitate was collected by vacuum filtration, washed three times with deionized water, and lyophilized to fine white powder.

(46) Synthesis of PLP Derivatives

(47) NDA, HDA, TDA or ODA was conjugated onto the PLP backbone at various degrees of substitution via DCC/DMAP coupling. Briefly, PLP (3 g), DMAP (0.6 g, 20 wt % of PLP) were dissolved in anhydrous DMSO/DMF (1:3 v/v). NDA, HDA, TDA or ODA was dissolved in chloroform and then transferred to the reaction solution. DCC (3 molar equivalents of decylamine) in anhydrous DMF was added dropwise. The reaction was monitored by thin-layer chromatography (CHCl.sub.3:MeOH:trithylamine=8:2:0.2, using ninhydrin to visualise amine). Solid impurities were removed by vacuum filtration and the reaction solution was added with 5 wt % NaOH in anhydrous ethanol and precipitated rapidly into five volumes of diethyl ether. The precipitate was collected and re-dissolved in deionized water. 0.2 M HCl solution was added to the solution to precipitate the polymer precipitate out. It was collected by vacuum filtration and re-dissolved in deionized water with 0.2 M NaOH. The precipitation-filtration-redissolution process was carried out twice to remove inorganic salts and residual organic solvents. The polymer was further purified by dialysis against deionized water in a Visking dialysis tubing (Medicell, MWCO 12-14 kDa). After dialysis, the polymer solution was titrated to pH 7.4 using 0.2 M NaOH and then lyophilized. To prepare acidic form, the dialysed polymer solution was acidified to around pH 3.0 using 0.2 M HCl. The precipitate was collected and lyophilized.

(48) The Mw of PLP (35.7 kDa) was determined an aqueous gel permeation chromatography (GPC) system. That means value of the degree of polymerization (n) is ˜130. The degree of substitution of each polymer was determined by .sup.1H-NMR spectroscopy in d.sub.6-DMSO (FIG. 1A). The ratio of the integral 0.77-0.91 ppm to the integral 7.45-7.64 ppm was used to calculate the degree of substitution. PLP-NDA 3%, 10% and 18% are expressed as the numbers of NDA grafts per 100 carboxylic acid groups along the parent backbone (mol %). The degree of substitution and the Mw of PLP were then used to calculate the molecular weight of the derivative containing hydrophobic pendant chains.

(49) Further PLP derivates can be obtained by reacting chemical compounds of table 4 with the PLP backbone via DCC/DMAP coupling as described above.

(50) TABLE-US-00005 TABLE 4 Example Chemical of formula Moeity HNR.sup.1R.sup.2 Supplier Catalogue number R.sup.1 = Alkyl chain short; Decylamine Sigma D2404 R.sup.2 = H R.sup.1 = Alkyl chain medium Octadecylamine Sigma 74750 R.sup.2 = H R.sup.1 = R.sup.2 = Alkyl chain short Dihexylamine Sigma 131202 R.sup.1 = R.sup.2 = Alkyl chain medium Dioctadecylamine Sigma 42358 R.sup.1 = Alkenyl chain short 3-Butenylamine hydrochloride Sigma 597678 R.sup.2 = H R.sup.1 = Alkenyl chain short 5-Hexenylamine GFS 5529 R.sup.2 = H Chemicals R.sup.1 = Alkenyl chain short oct-3-en-1-amine Enamine BBV-42249046 R.sup.2 = H Store R.sup.1 = Alkenyl chain medium tetradec-3-en-1-amine Enamine BBV-42256359 R.sup.2 = H Store R.sup.1 = Alkenyl chain medium Oleylamine Sigma O7805 R.sup.2 = H R.sup.1 = R.sup.2 = Alkenyl chain short ethyl(prop-2-en-1-yl)amine MolPort MolPort-000-005-271 R.sup.1 = R.sup.2 = Alkenyl chain medium bis[(2Z)-3-chlorobut-2-en-1- MolPort MolPort-000-163-345 yl]amine R.sup.1 = Alkenyl chain with multiple methyl[7-(methylimino)hepta- Enamine FCH4099593 C═C groups 1,3,5-trien-1-yl]amine Store R.sup.2 = Alkyl chain short R.sup.1 = Alkynyl chain short but-3-yn-1-amine MolPort MolPort-004-968-587 R.sup.2 = H hydrochloride R.sup.1 = Alkynyl chain short Hex-5-ynylamine Activate AS74680 R.sup.2 = H Scientific R.sup.1 = Alkynyl chain short oct-3-yn-1-amine Enamine FCH935266 R.sup.2 = H Store R.sup.1 = Alkynyl chain medium dodec-3-yn-1-amine Enamine FCH1282159 R.sup.2 = H Store R.sup.1 = Alkynyl chain medium 3-fluoro-2-methyloct-7-yn-1- EnamineStore BBV-70832810 R.sup.2 = H amine R.sup.1 = Alkynyl chain medium [4,4-dimethyl-2-(pent-4-yn-1- EnamineStore BBV-49722550 R.sup.2 = H yl)cyclohexyl]methanamine R.sup.1 = R.sup.2 = Alkynyl chain short bis(but-2-yn-1-yl)amine MolPort MolPort-001-991-305 R.sup.1 = Alkynyl chain medium (dec-1-yn-4-yl)(propyl)amine Chemspace CSC013776799 R.sup.2 = Alkyl chain short EXAMPLES OF SUSTITUTIONS R1 and R2 are aryls N-(2-Naphthyl)-1- Sigma 762660 naphthylamine R.sup.1 = Heteroaryl c-(2-p-Tolyl-imidazo[1,2- Sigma CDS008330 R.sup.2 = H a]pyridin-3-yl)-methylamine R.sup.1 = Alkyl substituted with aryl 1,1-bis(4-chlorophenyl)-2-[(2- Sigma CDS018870 and OH; fluorobenzyl)amino]-1-ethanol R.sup.2 = alkyl substituted with aryl R.sup.1 = aryl substituted with alkyl 4-Tetradecylaniline Sigma 233552 R.sup.2 = H R.sup.1 = R.sup.2 = alkyl substituted Bis[2-(di-tert- Sigma 739022 with PR.sup.3R.sup.4 butyphosphino)ethyl]amine solution R.sup.1 = alkyl substituted with aryl h-cys(trt)-nh2 Sigma CDS018559 R.sup.2 = alkyl substituted with SH and C(O)NH.sub.2 R.sup.1 and R.sup.2 together form a 1,7-Dibenzyl-1,4,7,10- Sigma CDS001040 heterocyclic ring optionally tetraazacyclododecane containing further heteroatoms Cyclic, amide and ether 2-(3-oxo-decahydro- Sigma CDS018799 quinoxalin-2-yl)-N-(4-phenoxy- phenyl)-acetamide thioether 2-[(2-amino-4- Sigma CDS015863 methylphenyl)sulfanyl]-N-(2- methylphenyl)acetamide R.sup.1 = alkyl substituted with aryl 4-Nitrophenethylamine Sigma 184802 R.sup.2 = H hydrochloride

(51) Turbidimetry

(52) The optical densities of polymer solutions at different pHs were measured on a UV-Vis spectrophotometer (Genesys 10S UV-Vis, Thermo Scientific, UK) at 480 nm. Polymer solutions were prepared with buffers at different pHs and equilibrated for 48 h.

(53) Fluorescence Spectroscopy

(54) Pyrene has been used as a probe to investigate the conformational transition of polymers in aqueous solution. 1.0 mM pyrene solution in absolute methanol was freshly prepared and added to each aqueous polymer solution to give a final pyrene concentration of 6×10.sup.−7 M. The polymer solutions were equilibrated for 48 h with protection from light. The excitation intensities at wavelengths of 338 and 333 nm (λ.sub.em=390 nm) of pyrene dissolved in the polymer solution were recorded on a spectrofluorometer (FluoroMax, HORIBA, Japan). The fluorescence intensity ratio of I.sub.338/I.sub.333 was calculated. The conformational transition in response to pH and concentration and the critical aggregation concentration (CAC) were then determined.

(55) Dynamic Light Scattering

(56) The hydrodynamic diameter and the size distribution of the polymers in aqueous solution were investigated using dynamic light scattering (Zetasizer Nano S. Malvern, UK). The polymer solutions were prepared in buffer at specific pHs and equilibrated for 48 h. All the samples were filtered through the 0.45-μm filter, and size measurements were conducted in 10-mm diameter cells at a scattering angle of 137°, repeated for 11 times for each run.

(57) Hemolysis

(58) The lipid membrane activity of the polymers was examined using the haemolysis assay of defibrinated sheep red blood cells (RBCs). Briefly, the polymers were added into 0.1 M phosphate buffer or 0.1 M citric buffer at specific pHs. RBCs were washed at least three times with 150 mM NaCl and resuspended in the polymer solution to a final concentration of 1-2×10.sup.8 RBCs mL.sup.−1. The negative control (without the presence of polymer) and the positive control (RBCs lysed in deionized water) were prepared with the same cell density. The samples were incubated in a shaking water bath (120 rpm) at 37° C. for a specific period, and then centrifuged at 4000 rpm for 4 min. The haemoglobin release was investigated by measuring the absorbance of the supernatant at 540 nm using the UV-Vis spectrophotometer. The relative haemolysis percentage was calculated using the following equation:
Haemolysis (%)=[(Sample absorbance−Negative control absorbance)/(Positive control absorbance−Negative control absorbance)]×100

(59) Cell Culture

(60) HeLa adherent epithelial cells (human cervical cancer cells) and A549 adherent epithelial cells (human lung cancer cells) were grown in DMEM supplemented with 10% (v/v) FBS and 100 U mL.sup.−1 penicillin unless specified otherwise. CHO adherent epithelial cells (Chinese hamster ovary cells) were cultured in DMEM supplemented with 1% (v/v) non-essential amino acids, 10% (v/v) FBS and 100 U mL.sup.−1 penicillin unless specified otherwise. The HeLa, A549 and CHO cells were trypsinized using trypsin-EDTA and maintained in a humidified incubator with 5% CO2 at 37° C.

(61) MES-SA adherent epithelial cells (human uterus cancer cells) and the corresponding multi-drug resistant cells MES-SA/DX5 were cultured in McCoy's 5a medium containing 10% (v/v) FBS and 100 U mL.sup.−1 penicillin unless specified otherwise. The MES-SA and MES-SA/DX5 cells were subcultured with EDTA solution (0.8 mM disodium EDTA, 68.5 mM NaCl, 6.7 mM sodium bicarbonate, 5.6 mM glucose and 5.4 mM KCl) and maintained in a humidified incubator with 5% CO2 at 37° C.

(62) SU-DHL-8 suspension B lymphocyte cells (human lymph node cells) were grown in RPMI-1640 medium supplemented with 10% (v/v) FBS and 100 U mL.sup.−1 penicillin and kept in a humidified incubator with 5% CO2 at 37° C.

(63) Mesenchymal stem cells (hMSCs, human bone marrow derived) were cultured in minimum essential medium Eagle containing 10% FBS and 100 U mL.sup.−1 penicillin. The cells were trypsinized using trypsin-EDTA and maintained in a humidified incubator with 5% CO2 at 37° C.

(64) Alamar Blue Assay

(65) The cytotoxicity of the polymers was evaluated using AlamarBlue® assay. Cells were seeded into 96-well plates (Corning, USA) containing culture medium (0.1 mL per well) at a density of 1×10.sup.4 cells/well for 24 h. The spent medium was replaced with 0.1 mL of sample solution containing 0.22 μm filter-sterilized polymer at various concentrations. After incubation for a specific period, the polymer-containing medium was replaced with DMEM containing 10% (v/v) AlamarBlue®. The plate was further incubated for 4 h according to the manufacturer's instructions and the fluorescence of each well was then measured by a spectrofluorometer (GloMax®-Multi Detection System, Promega) at emission wavelength of 580-640 nm with the excitation wavelength of 525 nm. The cytotoxic effect was determined from the fluorescence readings.

(66) Laser Scanning Confocal Microscopy

(67) Calcein, a membrane-impermeable fluorophore, was employed to assess the ability of the polymers to release endocytosed materials into the cytoplasm. 2 mL of HeLa, CHO or A549 cells (2×10.sup.5 cells mL-1) were seeded in glass-bottom culture dish (35 mm, MatTek, USA) and cultured in an incubator with 5% CO2 at 37° C. After 24 h, the spent medium was removed and replaced with 2 mL of 0.22 μm filter-sterilized serum-free medium containing the polymer at 0.5 mg mL.sup.−1 and calcein at 2 mg mL.sup.−1. In a control experiment, the cells were incubated with 2 mg mL.sup.−1 calcein alone. After incubation for a certain period, the cells were washed three times with D-PBS buffer replenished with medium. The cells were imaged by the laser scanning confocal microscope. Calcein was excited using a 488 nm laser and the emission was collected at 535 nm.

(68) The ability of the pseudopeptidic polymers to deliver a wide size range of FITC-labelled dextran (4-2000 kDa) was tested in 7 different cell liens. Adherent cells were cultured in a glass-bottom dish at a total cell number of 2×10.sup.5 cells for overnight followed by treatment with PBS containing the pseudopeptidic polymer and FITC-dextran at specific concentrations and pHs for a certain period of time. The cells were then washed with D-PBS and stained with LysoTracker and Hoechst and imaged as described above. For the suspension cell (SU-DHL-8), the cells were centrifuged and resuspended with 1 mL of PBS buffer containing the pseudopeptidic polymer and FITC-dextran to reach a final cell concentration of 4×10.sup.5 cells mL.sup.−1. After 30 min of incubation, the cells were centrifuged to remove the supernatant and resuspended with D-PBS for three times and stained with LysoTracker and Hoechst before imaging.

(69) Flow Cytometry

(70) Flow cytometry was employed to quantitatively evaluate the polymer mediated payload delivery. Adherent cells were cultured in a 6-well plate (3×10.sup.5 cells per well) for overnight and treated with 1 mL PBS containing the pseudopeptides and FITC-dextran at specific concentrations for a certain period of time. Afterwards, the cells were washed with D-PBS for three times, detached using trypsin and then centrifuged at 1000 rpm. The cell pellet was resuspended in serum free culture medium and filtered through 40 μm Flowmi™ tip strainers (Bel-Art, USA) to remove cell aggregates. SU-DHL-8 suspension cells (8×10.sup.5 cell mL.sup.−1) were treated with PBS containing the polymers and FITC-dextran for 30 min. The cells were then centrifuged to remove the supernatant and filtered. The flow cytometry was carried out with an LSRFortessa cell analyzer at excitation wavelength of 488 nm.

(71) Intracellular Sugar Delivery

(72) 306 mOsm phosphate buffered saline (PBS, pH=7.4) was prepared by dissolving 136.89 mM sodium chloride, 8.10 mM sodium phosphate dibasic heptahydrate, 2.68 mM potassium chloride and 1.47 mM potassium dihydrogen orthophosphate into 1 L de-ionized water. 660 mOsm PBS buffer was made by dissolving 297.74 mM NaCl, 17.62 mM Na.sub.2HPO.sub.4.7H.sub.2O, 5.83 mM KCl and 3.20 mM KH.sub.2PO.sub.4 into 1 L deionized water.

(73) Sugar solutions (with or without polymer) were prepared by dissolving a certain amount of sugar into PBS buffer, and their pH was adjusted to a desired value. The anthrone solution was prepared by adding 125 mg of anthrone into 66% (v/v) H.sub.2SO.sub.4.

(74) Sheep red blood cells were centrifuged at approximately 1200 rcf for 4 minutes. 306 mOsm PBS buffer was then added after the removal of the supernatant. This process was repeated 3 times in order to completely remove free haemoglobin. The sugar solution was added to the pre-washed RBCs achieve the 15% cell packed volume (CPV). Cells were homogeneously re-suspended into solution. Subsequently, RBCs were incubated for a certain period of time at a desired temperature.

(75) After incubation, haemolysis was examined via the measurement of the absorbance of the supernatant at 541 nm by UV-Vis spectrophotometer. Cell pellets were washed by the iso-osmotic PBS buffer (660 mOsm) twice to remove the extracellular sugar molecules. RBCs were then lysed by 80% methanol in an 85° C. water bath for 1 hour and centrifuged at approximately 11000 rcf for 4 minutes. The supernatant was placed in an oven at 100° C. for overnight to completely remove water and methanol, followed by the addition of 2 mL deionized water. The anthrone method was then carried out to quantify the intracellular sugar concentration. Specifically, 0.5 mL of the above solution was added into the anthrone solution followed by water bath at 100° C. for the 15 minutes. Then, absorbance was measured by UV-Vis at 620 nm wavelength to calculate the amount of sugar molecules loaded into the cell interior.

(76) Sheep red blood cells (RBCs) with a packed volume of 15% (3.5×10.sup.9 RBCs per ml) were placed in a 2-mL centrifuge tube, to which was added the PBS buffer solution at the desired pH containing specific concentrations of polymer and trehalose. After incubation at 37° C. for a certain period of time, the amount of trehalose loaded into the cell interior was determined using the method described above. The blood solution was then transferred to a 2-ml polypropylene cryovial. The cryovial tubes were immersed into liquid nitrogen (−196° C.) for a certain period of time. RBCs were then thawed in a 37° C. water bath for 15 min. The cell suspension was then centrifuged and the cell pellet was lysed to calculate the amount of viable cells from the absorbance of the lysed RBC solution at 541 nm. Haemolysis during the sugar delivery and the freezing-thawing processes was also measured and used to calculate the cryosurvial rate.

(77) Confocal Microscopy and Flow Cytometry of RBCs

(78) Membrane-impermeable calcein was applied to further investigate the polymer-mediated delivery into erythrocytes. RBCs (15% packed cell volume) were washed three times and incubated with PBS buffer containing 0.36 M trehalose+0.1 mM calcein, 0.36 M trehalose+0.1 mM calcein+0.8 mg mL.sup.−1 PP50, or 0.36 M trehalose+0.1 mM calcein+0.8 mg mL.sup.−1 PLP-NDA 18%, respectively at pH 6.1 in a 37° C. shaking water bath (120 rpm) for 15 min. After incubation, the samples were washed twice with PBS buffer by centrifugation and imaged with an LSM-510 inverted laser scanning confocal microscope (Zeiss, Germany) at 37° C. Calcein was excited at 488 nm, and the emission at 535 nm was collected. The flow cytometry measurements were carried out using an LSRFortessa cell analyzer (BD, USA) with an excitation wavelength of 488 nm.

(79) To evaluate the membrane permeability after trehalose loading and washing, RBCs were incubated with trehalose in the absence or in the presence of polymer at pH 6.1 at 37° C. for 15 min, followed by washing twice with pH 7.4 PBS buffer. Then the processed RBCs were incubated with pH 7.4 PBS buffer containing 1 μM calcein and imaged with the laser scanning confocal microscope as described before.

(80) Atomic Force Microscopy (AFM)

(81) To further study the mechanism of rapid trehalose intracellular loading mediated by PLP-NDA 18%, AFM was applied to exam the polymer-cell interaction. RBCs were washed three times, incubated with PBS buffer containing 0.36 M trehalose, 0.36 M trehalose+0.8 mg mL.sup.−1 PP50, or 0.36 M trehalose+0.8 mg mL-1 PLP-NDA 18% at pH 6.1 for 15 min, and immobilized on a polylysine-coated microscope slide. The cells were crossed linked in glutaraldehyde (1%) for 10 min, washed and then air dried. AFM was performed using the Asylum MFP-3D microscope (Oxford instruments Asylum Research, US) in tapping mode. Nanosensors PPP-NCHR tips with resonant frequency around 320 kHz, tip radius 7 nm and spring constant 42 N m.sup.−1 were used and tuned to a target tapping amplitude of 1-2 V.

EXAMPLE 1

(82) Formation of PLP grafted with NDA (C10) and other pendant hydrophobic chains including HDA (C7), TDA (C14) and ODA (C18) has been confirmed using 1H-NMR and FTIR spectra (FIG. 1).

EXAMPLE 2

(83) pH dependent transmittance (FIG. 2A) and pH- and concentration-dependent hydrophobic association (FIGS. 2B & 2C) of the aqueous solutions of the PLP substituted with different degrees of NDA (%). As shown PLP-NDA displayed pH-dependent properties. The increase in pH at the onset of precipitation (pH.sub.p) and hydrophobic association (pH.sub.h), the widening of pH range for association and the decrease in CAC were achieved by grafting PLP with hydrophobic pendant side chains and increasing the degree of substitution (Table 1).

EXAMPLE 3

(84) pH and concentration dependent particle size distribution of the polymers PLP and PLP-NDA (FIG. 3, Tables 2 & 3). The increase in the degree of substitution with hydrophobic pendant chains can lead to the decrease in particle size due to stronger hydrophobic interactions.

EXAMPLE 4

(85) Haemolysis of red blood cells treated with PLP and its derivatives was found to be dependent on pH, concentration and incubation time (FIG. 4). The membrane activity of the polymers can be manipulated by the type of hydrophobic side chains and the degree of substitution. At pH 7.4 the level of haemolysis was found to be low or negligible, whilst upon acidification the membrane-destabilizing capacity was increased. The increase of the degree of substitution up to 18% of hydrophobic pendant chains led to enhanced membrane activity.

EXAMPLE 5

(86) In vitro cytotoxicity of PLP-NDA 18% was tested in HeLa cells (FIG. 5A), CHO cells (FIG. 5B) and A549 cells (FIG. 5C) and at various PLP-NDA concentrations for 4 h (blank), 12 h (grey), 24 h (black) and 48 h (stripped). The IC.sub.50 of PLP-NDA 18% against HeLa for 12 h of treatment was 2.94±0.28 mg mL.sup.−1, while it decreased to 1.59±0.22 mg mL.sup.−1 and 1.05±0.14 mg mL.sup.−1 after treatment for 24 h and 48 h respectively. CHO cells demonstrated the better tolerance than HeLa cells, with IC.sub.50 of 2.81±0.95 mg mL.sup.−1 for 48 h treatment, while A549 showed the highest IC.sub.50 of 4.51±0.06 mg mL.sup.−1 for 48 h treatment. FIG. 5D shows that the cell viability of different types of cells treated with 0.5 mg mL-1 of PLP or PLP-NDA 18% for 24 h was not significantly different.

EXAMPLE 6

(87) Hela (FIG. 6A), CHO (FIG. 6B) and A549 cells (FIG. 6C) treated with PLP-NDA 18% and membrane-impermeable calcein showed an increased fluorescent signal due to the release of endocytosed calcein into the cytoplasm when compared to cells treated with calcein alone or treated with PLP and calcein.

EXAMPLE 7

(88) PLP-NDA 18% aided delivery of FITC-dextran with different molecular weights (Mw) into HeLa cells after incubation at pH 6.5 for 30 min (FIG. 7A). No significant transport was detected when cells were incubated with FITC-dextran only (FIG. 7B) or with PLP-NDA 18% and FITC dextran at pH 7.4 (FIG. 7C).

EXAMPLE 8

(89) Efficient transport was dependent on the polymer concentration (FIG. 8) and was highest at the PLP-NDA 18% concentration of 0.5-2 mg mL.sup.−1.

EXAMPLE 9

(90) Derivatives of PLP containing alkyl chains with different lengths facilitated FITC-dextran delivery (FIG. 9). The delivery efficiency was ranked in the order of PLP-NDA 18%>PLP-TDA 18%>PLP-HDA 18%>PLP-ODA 18%. No significant intracellular delivery was observed when the cells were incubated with FITC-dextran only at pH 6.5.

EXAMPLE 10

(91) PLP-NDA 18% mediated intracellular payload delivery in response to extracellular pH, as demonstrated by confocal microscopy (FIG. 10) and flow cytometry (FIG. 11). Limited intracellular delivery of FITC-dextran was detected when the cells were co-incubated with PLP-NDA 18% and FITC-dextran at pH 7.4. The delivery efficiency considerably increased with decreasing pH. The highest delivery efficiency was achieved at extracellular pH 5.5-6.5.

EXAMPLE 11

(92) Confocal microscopy (FIG. 12) and flow cytometry (FIG. 13) measurements showed that, when HeLa cells were co-incubated with PLP-NDA 18% and FITC-dextran at pH 6.5, intracellular delivery started only after 10 min of treatment. The delivery efficiency increased with the extension of incubation time.

EXAMPLE 12

(93) The wide applicability of PLP-NDA 18% mediated intracellular delivery was demonstrated via confocal microscopy (FIG. 14) and flow cytometry measurements (FIG. 15). Strong diffuse green fluorescence was observed in all cell types tested, including HeLa adherent epithelial cells (human cervical cancer cells), CHO adherent epithelial cells (Chinese hamster ovary cells), A549 adherent epithelial cells (human lung cancer cells), SU-DHL-8 suspension B lymphocyte cell line (human lymph node cells), MES-SA adherent epithelial cell line (human uterus cancer cells), MES-SA/DX5 adherent multi-drug resistant cell line and human mesenchymal stem cells (hMSCs, human bone marrow derived). This confirms that the comb-like polymer can efficiently deliver macromolecules into a variety of cell types, including adherent and suspension cells, cancerous and non-cancerous cells, multidrug resistant cells, lymphocytes and stem cells.

EXAMPLE 13

(94) In vitro cytotoxicity of PLP-NDA 18% against different cell types at various extracellular pHs, polymer concentrations and time durations was evaluated (FIG. 16). The polymer was well tolerated by a variety of cell types within a wide polymer concentration range after various durations of incaution at both acidic and neutral pHs.

EXAMPLE 14

(95) Trehalose loading and haemolysis of erythrocytes was monitored at different conditions such as incubation time and polymer concentration. Increase of the intracellular trehalose concentration could be measured after 15 min incubation with PLP-NDA 18% (300-800 μg mL.sup.−1) at pH 7.05. An intracellular trehalose concentration of 215 mM was achieved after cells were incubated in 800 μg mL.sup.−1 PLP-NDA 18% for 60 min (FIG. 17). Haemolysis of erythrocytes was found to be increased relative to the PLP-NDA polymer concentration (FIG. 18).

EXAMPLE 15

(96) Trehalose uptake and haemolysis is pH dependent. Uptake was increased after 15 min of treatment at a pH between 6.1 and 5.6 and haemolysis was low for all concentrations tested (FIG. 19). Haemolysis at this pH was reduced when cells were treated with 600 μg mL.sup.−1 PLP-NDA (FIG. 20).

EXAMPLE 16

(97) Trehalose loading and haemolysis is dependent on PLP-NDA incubation time (FIG. 21) and temperature (FIG. 22). The intracellular trehalose concentration in erythrocytes in 0.36 M trehalose solution and with addition of 800 μg mL.sup.−1 PLP-NDA 18% at pH 6.1 reached a high level up to approximately 0.3 M after 60 min of loading, with haemolysis below 30%. Among the temperature range (31-40° C.) tested, the intracellular trehalose and haemolysis peaked at 37° C.

EXAMPLE 17

(98) Trehalose loading and haemolysis is dependent on the extracellular trehalose concentration. Incubation of cells in a solution containing 0.36 M trehalose and 800 μg mL.sup.−1 PLP-NDA 18% for 1 h resulted in haemolysis of below 30% and a high intracellular trehalose concentration of 300 mM (FIG. 23).

EXAMPLE 18

(99) The impact of the length of hydrophobic pendant chains and the degree of substitution on trehalose loading (FIGS. 24 and 26) and haemolysis (FIGS. 25-26) of cells was monitored. The PLP substituted with NDA (C7) at the 18% degree of substitution showed the optimal intracellular trehalose loading.

EXAMPLE 19

(100) The membrane-impermeable dye calcein was mixed with trehalose to trace its translocation into cells by confocal microscopy (FIG. 27) and flow cytometry (FIG. 28). As shown PLP-NDA 18% induced intracellular delivery of payloads (calcein and trehalose) after only 15 min of treatment, much more rapidly than PP50 (PLP grafted with hydrophobic amino acid phenylalamine) which has been reported to take as long as 9 hours. PLP-NDA 18% induced intracellular delivery was also considerably more efficient than PP50. At pH 7.4, both polymers mediated limited intracellular delivery.

EXAMPLE 20

(101) Membrane integrity of the erythrocytes post sugar loading was evaluated (FIG. 29). The erythrocyte membrane remained impermeable after sugar loading in the absence or in the presence of PLP-NDA 18%, as calcein was not able to permeable through cell membrane after washing and pH adjustment to 7.4. This suggests that the polymer didn't cause permanent membrane damage and the polymer mediated membrane permeabilization was reversible.

EXAMPLE 21

(102) The membrane surface roughness of erythrocytes mediated by different polymers was compared using topographic AFM (FIG. 30, Table 5). The cell surface roughness was ranked in the order of PLP-NDA 18%>PP50>control, suggesting that PLP-NDA 18% facilitated much stronger interaction with cell membrane compared to PP50

EXAMPLE 22

(103) The erythrocytes (FIG. 31) treated with 800 μg mL.sup.−1 PLP-NDA 18% and 0.36 M trehalose solution at pH 6.1 for 15 min can reach an approximately survival of approximately 85%, significantly higher than the cells treated with 0.36 trehalose solution only. The change of cryosurvival was not significant when the duration of cryopreservation was extended from 5 min to 168 hours.