Patent Application
20170037184
The present invention relates to vitamin E-based amphiphilic copolymers and compositions containing vitamin E-based amphiphilic copolymers and nanocarriers. The present invention also relates to methods of making vitamin E-based amphiphilic copolymers and nanocarriers, and methods of using vitamin E-based amphiphilic copolymers and nanocarriers, for example, as a drug delivery device.
Polymeric micelles are versatile drug carrier systems that demonstrate great promise in the delivery of anticancer drugs. Currently, there are six micellar formulations for paclitaxel, doxorubicin and platinum drugs undergoing clinical trials and many more being explored in the preclinical studies. Composed of diverse amphiphilic copolymers, polymeric micelles are self-assembled supramolecular aggregates displaying typical core-shell architecture.
For example, D-α-Tocopherol polyethylene glycol succinate 1000 (TPGS1K) is an amphiphilic copolymer generated by coupling PEG 1000 (PEG1K) to Vitamin E (VE) succinate via an ester linkage. A safe pharmaceutical excipient approved by the FDA, TPGS1K has been extensively investigated as an emulsifier, solubilizer and stabilizer in a variety of nano-sized drug delivery systems such as nanoparticles and micelles. With high lipophilicity conferred by the aliphatic chain and aromatic ring of the VE moiety, the inclusion of TPGS1K in the mixed micelles not only greatly increases the loading capacity for hydrophobic drugs, but also stabilizes the micellar core by reducing the molecular motion of the copolymers. Nevertheless, as a micelle forming copolymer itself, TPGS1K has an unfavorably high critical micelle concentration (CMC) at 0.2 g/L.
In a recent study, PEG was extended to 2 KDa and conjugated with VE at a 1:2 molar ratio, resulting in a PEG2K-VE2 copolymer, which formed micelles at a considerably lower CMC (1.14 mg/L), and could deliver doxorubicin with more potent anticancer efficacy than doxorubicin in the free form or in the TPGS1K micelles. In another recent study, it was demonstrated that by further increasing PEG size to 5 KDa, the PEG5K-VE2 micelles (CMC≈1.8 mg/L) functioned as an improved formulation for paclitaxel and achieved stronger tumor growth inhibition than Taxol® and paclitaxel-loaded PEG2K-VE2 micelles.
MicroRNAs (miRNAs) are endogenous small non-coding RNAs (˜22 nucleotides in length) that control gene expression at the posttranscriptional level by either inducing translation repression or promoting the degradation of messenger RNAs (mRNAs). By imperfectly basepairing with the 3′-untranslated region of the target mRNAs, each individual miRNA can concomitantly targets tens to hundreds of gene transcripts, regulating a large network of signaling pathways in the cells. It has been estimated that over 60% of all protein-coding genes are subject to miRNA regulation. As such, miRNAs are master regulators of gene expression essential to all aspects of the cellular processes.
Dysregulated miRNAs play causal roles in the onset, development and metastasis of cancer. Certain miRNAs have been recognized to function as tumor suppressors by directly downregulating oncogenes, and the expression such tumor suppressor miRNAs are commonly lost in tumor cells. MiRNA replacement therapy aims to restore tumor suppressor miRNAs in tumor cells to achieve anticancer effect. To reconstitute intracellular miRNA levels, one prevailing strategy is to employ miRNA mimics, which are synthetic double-stranded RNAs that contain the mature miRNA strands identical to the endogenous miRNAs. The first miRNA replacement therapy-MRX34-miR-34a mimic delivered by liposomal nanoparticles, has entered Phase I clinical trials for the treatment of hepatocellular carcinoma since 2013.
Let-7 is a family of tumor suppressor miRNAs crucially involved in cell differentiation, proliferation and motility by directly targeting pleotropic oncogenes including RAS, MYC, HMGA2, GAB2 and FN1 among others. Abundantly expressed in the healthy tissues, let-7 is frequently suppressed in solid tumors and is associated with poor prognosis in non-small cell lung cancer (NSCLC), breast and gastric cancers. Repletion of let-7 has been shown to suppress tumor initiation in the mouse models of NSCLC. However, delivery of let-7 mimic or let-7-encoding plasmids to the pre-established tumors exhibits limited anticancer potency owing to the inability of let-7 to trigger apoptosis in tumor cells.
Efforts continue to further develop effective drug carrier systems capable of effectively delivering drugs within a patient, such as anticancer drugs to a tumor location within a human or other animal.
The present invention addresses some of the difficulties and problems discussed above by the discovery of new amphiphilic copolymers and nanocarriers formed therefrom, both of which have exceptional drug association/complexing properties, as well as complex stability.
Accordingly, in one exemplary embodiment, the present invention is directed to amphiphilic copolymers. In one exemplary embodiment, the amphiphilic copolymers of the present invention comprise: polyethylene glycol, and three or more vitamin E units bonded (i.e., covalently bonded directly or indirectly) to the polyethylene glycol. In another exemplary embodiment, the amphiphilic copolymers of the present invention comprise a crosslinked amphiphilic copolymer comprising: polyethylene glycol, and two or more vitamin E units bonded (i.e., covalently bonded directly or indirectly) to the polyethylene glycol. In some embodiments, the amphiphilic copolymer comprises three or more vitamin E units bonded (directly or indirectly) to the polyethylene glycol. Typically, the amphiphilic copolymer comprises from four to eight vitamin E units bonded to the polyethylene glycol.
Each vitamin E unit may be bonded to the polyethylene glycol via one or more divalent linkages. Typically, the one or more divalent linkages comprise one or more divalent linkages covalently bonded between each vitamin E unit and the polyethylene glycol. In addition to the one or more divalent linkages, the amphiphilic copolymers (and nanocarriers formed therefrom) of the present invention may further comprise one or more crosslinking moieties bonded to the polyethylene glycol and/or the one or more divalent linkages.
The present invention is further directed to compositions comprising the herein-described amphiphilic copolymers and/or nanocarriers. In one exemplary embodiment, the composition of the present invention comprises one or more amphiphilic copolymers, as described herein (or nanocarriers formed therefrom), and at least one biologically active substance incorporated within the one or more amphiphilic copolymers (or nanocarriers formed therefrom). Suitable biologically active substances include, but are not limited to, a drug, an anti-carcinogenic compound, a nucleic acid, another small molecule, or any combination thereof. In one desired embodiment, the biologically active substance comprises an anti-carcinogenic compound. Suitable anti-carcinogenic compounds include, but are not limited to, paclitaxel, docetaxel, carbazitaxel, ixabepilone, eribulin, topotecan, irinotecan, SN-38, doxorubicin, daunorubicin, idarubicin, epirubicin, etoposide and omacetaxine. Suitable nucleic acids include, but are not limited to, miRNA mimics (e.g., let-7 mimic, and miR-34 mimic), anti-miRs, siRNAs and antisense oligonucleotides. The compositions may further comprise one or more additional components including, but not limited to, combinations of two or more of the herein-described nanocarriers, water, sodium phosphate, sodium chloride, glucose, HEPES (i.e., 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) and mannitol.
The present invention is even further directed to methods of making amphiphilic copolymers, nanocarriers and compositions containing the same. In one exemplary embodiment, the method of making an amphiphilic copolymer comprises covalently bonding three or more vitamin E units to polyethylene glycol. The methods of making amphiphilic copolymers, nanocarriers, or compositions containing the same, may further comprise one or more steps including, but not limited to, reacting a functionalized polyethylene glycol having a terminal amine end group and a terminal carboxyl end group with one or more divalent linkages; reacting functional groups on the one or more divalent linkages with the three or more vitamin E units; reacting one or more crosslinking moieties with either the polyethylene glycol, the one or more divalent linkages, or both; combining at least one biologically active substance with the amphiphilic copolymer or nanocarrier; and incorporating (i) the biologically active substance and (ii) the amphiphilic copolymer and/or nanocarrier into an aqueous solution.
The present invention is even further directed to a method of delivering one or more drugs to a patient, wherein the method comprises administering an effective amount of any one of the herein-described amphiphilic copolymers, nanocarriers or compositions to the patient.
These and other features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.
To promote an understanding of the principles of the present invention, descriptions of specific embodiments of the invention follow and specific language is used to describe the specific embodiments. It will nevertheless be understood that no limitation of the scope of the invention is intended by the use of specific language. Alterations, further modifications, and such further applications of the principles of the present invention discussed are contemplated as would normally occur to one ordinarily skilled in the art to which the invention pertains.
The present invention is directed to amphiphilic copolymers and nanocarriers comprising a reaction product of PEG and vitamin E, as well as compositions containing the same. The present invention is further directed to methods of making amphiphilic copolymers, nanocarriers and compositions containing a reaction product of PEG and Vitamin E. The present invention is even further directed to methods of using the amphiphilic copolymers, nanocarriers and compositions in various applications including, for example, as drug delivery devices.
The amphiphilic copolymers of the present invention utilize (i) polyethylene glycol (PEG) as a hydrophilic block, which shields the amphiphilic copolymer (and nanoconstructs formed therefrom) and confers a stealth property by sterically hindering protein adsorption, and (ii) a tailored hydrophobic segment in the form of multiple Vitamin E units (i.e., derived from α-tocopheryloxyacetic acid) to optimize encapsulation of a nanocarrier payload (e.g., a drug). The disclosed micellar nanocarriers enable a tunable loading capacity for various hydrophobic drugs within the highly lipophilic micellar core via physical entrapment, which alleviates the use of organic solvents and greatly increases the aqueous solubility of otherwise water-insoluble drugs.
Another potential advantage of the herein-disclosed polymeric micelles (i.e., nanocarriers) for cancer therapy resides in their nanoscale dimensions, which can allow for preferential accumulation of anticancer drugs in the tumor via the enhanced permeability and retention (EPR) effect owing to leaky vasculature and diminished lymphatic drainage in solid tumors. To realize this benefit, it is desirable that the nanocarrier micelles maintain structural integrity and retain the payload for sufficient duration while circulating in the bloodstream. Although thermodynamic and kinetic instability of micelles in vivo poses significant challenges to minimize the burst release of a given payload before the drug-loaded micelles arrive at the tumor site, the nanocarrier micelles of the present invention exhibit exceptional stability when tested in in vivo conditions (as discussed in the Example section below).
To reinforce structural integrity of the nanocarrier polymeric micelles of the present invention and assist these self-assembled aggregates so as to withstand the destabilizing environment in vivo, adjacent polymeric chains of the nanocarrier polymeric micelles may be coyalently crosslinked to generate a single, nanostructured macromolecule. Various chemical reactions, such as radical polymerization, UV crosslinking, disulfide linkage, polyelectrolyte complexation, silicon chemistry and click chemistry, may be used to form the crosslinked micelles. By conjugating the polymer backbone with cross-linkable moieties or introducing external bifunctional crosslinkers, cross-linkages may be formed at the shell, core or core-shell intermediate region of the herein-disclosed nanocarrier micelles. For the herein-disclosed crosslinked nanocarrier micelles to serve as biocompatible and biodegradable drug carriers, it is desirable for the crosslinking reactions to occur in an aqueous solution, employ/produce non-toxic substances, and be readily reversible in the tumor to facilitate the release of the payload and the elimination of the polymers.
In some embodiments, the amphiphilic copolymers and nanocarriers of the present invention utilize disulfide linkage crosslinking, which demonstrates excellent reversibility and biocompatibility. For example, cysteine-containing copolymers may be utilized to install free thiol groups onto the nanocarrier polymer chains, so that disulfide-crosslinked nanocarrier micelles can be generated under mildly oxidative conditions. Alternatively, external disulfide-containing linkers may be used to produce the crosslinked nanocarrier micelles. In other embodiments, thioctic acid (TA), an endogenous metabolite and a naturally occurring antioxidant, may be grafted onto the amphiphilic copolymer backbone as a crosslinker. The disulfide-containing thioctic ring is susceptible to ring opening polymerization in the presence of a catalytic amount of dithiothreitol (DTT), which results in the formation of linear polydisulfide crosslinks among adjacent polymer chains within the nanocarrier micelles. As delivery vehicles for hydrophobic chemotherapeutic drugs including paclitaxel, docetaxel and doxorubicin, the disulfide-crosslinked micelles demonstrate improved stability in the circulation and enhanced antitumor efficacy in murine xenograft models.
Further, disulfide crosslinks are redox-responsive and prone to cleavage through thiol-disulfide exchange reactions with glutathione (GSH), an endogenous reducing agent present at much higher level in the cytosol of tissue cells (1-10 mM) than in plasma and the interstitial fluid (2-10 μM). GSH level is found to be further elevated in the tumor cells, rendering disulfide-crosslinked nanocarrier micelles particularly suitable for intratumor delivery of anticancer drugs.
As discussed herein, amphiphilic copolymers and nanocarriers of the present invention comprise a reaction product of PEG and multiple Vitamin E units. Other amphiphilic copolymers and nanocarriers of the present invention comprise a reaction product of, inter alia, PEG, multiple Vitamin E units, one or more divalent linkages, and one or more moieties such as a crosslinking moiety or a cationic moeity. The amphiphilic copolymers and nanocarriers of the present invention are further described in the embodiments below.
Amphiphilic Copolymers, Nanocarriers and Compositions Embodiments:
1. An amphiphilic copolymer comprising: polyethylene glycol, and three or more vitamin E units bonded to the polyethylene glycol.
2. A crosslinking amphiphilic copolymer comprising: polyethylene glycol, and two or more vitamin E units bonded to the polyethylene glycol. As used herein, the term “crosslinking amphiphilic copolymer” refers to amphiphilic copolymers in which at least a portion of the amphiphilic polymer is crosslinkable with itself and/or other amphiphilic copolymers.
3. The amphiphilic copolymer of embodiment 2, wherein said amphiphilic copolymer comprises three or more vitamin E units bonded to the polyethylene glycol.
4. The amphiphilic copolymer of any one of embodiments 1 to 3, wherein said amphiphilic copolymer comprises from four to eight vitamin E units bonded to the polyethylene glycol. It should be understood that the term “said amphiphilic copolymer” or “the amphiphilic copolymer” used in the embodiments below refers to both the amphiphilic copolymer recited in embodiment 1 and the crosslinking amphiphilic copolymer recited in embodiment 2.
5. The amphiphilic copolymer of any one of embodiments 1 to 4, wherein said amphiphilic copolymer comprises four vitamin E units bonded to the polyethylene glycol.
6. The amphiphilic copolymer of any one of embodiments 1 to 5, wherein said polyethylene glycol has a molecular weight of from about 1000 to 500,000 (or any molecular weight between 1000 and 500,000, e.g., 5000, or any range of values between 1000 and 500,000, e.g., 4500-5500, in increments of 1.0).
7. The amphiphilic copolymer of any one of embodiments 1 to 6, wherein said polyethylene glycol has a molecular weight of from about 5000 to 20,000.
8. The amphiphilic copolymer of any one of embodiments 1 to 7, wherein each vitamin E unit is bonded to said polyethylene glycol via one or more divalent linkages.
9. The amphiphilic copolymer of any one of embodiments 1 to 8, said amphiphilic copolymer having a structure:
wherein PEG represents said polyethylene glycol, V represents a given vitamin E unit, K represents a first divalent linkage, and independently each n=0 to 5.
10. The amphiphilic copolymer of any one of embodiments 1 to 9, said amphiphilic copolymer having a structure:
11. The amphiphilic copolymer of any one of embodiments 1 to 8, said amphiphilic copolymer having a structure:
wherein PEG represents said polyethylene glycol, V represents a given vitamin E unit, K represents a first divalent linkage, R represents a crosslinking or cationic moiety, independently each m=0 to 5, and independently each n=0 to 20.
12. The amphiphilic copolymer of any one of embodiments 1 to 8 and 11, said amphiphilic copolymer having a structure:
13. The amphiphilic copolymer of any one of embodiments 1 to 8, said amphiphilic copolymer having a Structure′
wherein PEG represents said polyethylene glycol, V represents a given vitamin E unit, K represents a first divalent linkage, A represents a second divalent linkage, R represents a crosslinking or cationic moiety, independently each m=0 to 5, and independently each n=0 to 20.
14. The amphiphilic copolymer of any one of embodiments 1 to 8 and 13, said amphiphilic copolymer having a structure:
15. The amphiphilic copolymer of any one of embodiments 1 to 8, said amphiphilic copolymer having a structure:
wherein PEG represents said polyethylene glycol, V represents a given vitamin E unit, K represents a first divalent linkage, independently each m=0 to 5, and independently each n=0 to 20.
16. The amphiphilic copolymer of any one of embodiments 1 to 8 and 15, said amphiphilic copolymer having a structure:
17. The amphiphilic copolymer of any one of embodiments 1 to 8, said amphiphilic copolymer having a structure:
wherein PEG represents said polyethylene glycol, V represents a given vitamin E unit, K represents a first divalent linkage, A represents a second divalent linkage, PEI represents a polyetitylenimine unit, and independently each m=0 to 5.
18. The amphiphilic copolymer of any one of embodiments 1 to 8 and 17, said amphiphilic copolymer having a structure:
19. The amphiphilic copolymer of any one of embodiments 8 to 18, wherein each of said one or more divalent linkages independently comprises lysine, Nα-Fmoc-Nε-Boc-L-lysine, or aspartic acid.
20. The amphiphilic copolymer of any one of embodiments 8 to 19, wherein each K comprises lysine or Nα-Fmoc-Nε-Boc-L-lysine.
21. The amphiphilic copolymer of any one of embodiments 8 to 20, wherein each K comprises Nα-Fmoc-Nε-Boc-L-lysine.
22. The amphiphilic copolymer of any one of embodiments 13 to 14 and 17 to 21, wherein each A comprises aspartic acid or β-benzyl-L-aspartate N-carboxy anhydride.
23. The amphiphilic copolymer of any one of embodiments 13 to 14 and 17 to 22, wherein each A comprises β-benzyl-L-aspartate N-carboxy anhydride.
24. The amphiphilic copolymer of any one of embodiments 1 to 8, 13 and 19 to 23, said amphiphilic copolymer having a structure:
wherein x equals a number from 0 to 10.
25. The amphiphilic copolymer of any one of embodiments 9 to 24, wherein each V comprises α-tocopheryloxyacetic acid (i.e., each V comprises α-tocopheryloxyacetic acid minus an OH group).
26. The amphiphilic copolymer of any one of embodiments 1 to 25, wherein each vitamin E unit comprises α-tocopheryloxyacetic acid (i.e., each vitamin E unit comprises α-tocopheryloxyacetic acid minus an OH group).
27. The amphiphilic copolymer of any one of embodiments 11 to 14 and 19 to 26, wherein each R independently comprises thioctic acid, cysteine, diethylenetriamine, triethylenetetramine, tris(2-aminoethyl)amine or N,N-diisopropylethylenediamine (i.e., each R group represents a given R group minus one or more atoms removed from the R group so as to bond with the PEG polymer chain of the nanocarrier, e.g., an OH group for thioctic acid).
28. The amphiphilic copolymer of any one of embodiments 11 to 14 and 19 to 27, wherein each R independently comprises diethylenetriamine, triethylenetetramine, tris(2-aminoethyl)amine or N,N-diisopropylethylenediamine. In some preferred embodiments, each R comprises diethylenetriamine.
29. The amphiphilic copolymer of any one of embodiments 17 to 20, 22 and 25 to 27, wherein each PEI independently comprises a polyethylenimine unit having a molecular weight ranging from about 200 to about 2500 (or any value between 200 and 2500, e.g., 600, or any range of values between 200 and 2500, e.g., 600-650, in increments of 1.0).
30. The amphiphilic copolymer of any one of embodiments 9 to 29, wherein each PEG is represented by:
H3CO—(CH2CH2O)z—CH2CH2NH—
wherein z ranges from about 25 to 12,500 (or any value between 25 and 12,500, e.g., 115, or any range of values between 25 and 12,500, e.g., 100-137, in increments of 1.0).
31. The amphiphilic copolymer of embodiment 30, wherein z ranges from about 100 to about 125.
32. The amphiphilic copolymer of any one of embodiments 2 to 31, wherein said crosslinking amphiphilic copolymer comprises one or more crosslinking moieties bonded (directly or indirectly) to said polyethylene glycol, wherein each crosslinking moiety independently comprises thioctic acid and cysteine.
33. The amphiphilic copolymer of any one of embodiments 2 to 32, wherein said crosslinked amphiphilic copolymer comprises one or more crosslinking moieties bonded (directly or indirectly) to said polyethylene glycol, wherein each crosslinking moiety comprises thioctic acid.
34. A nanocarrier comprising one or more amphiphilic copolymers, wherein each amphiphilic copolymer independently comprises the amphiphilic copolymer of any one of embodiments 1 to 33.
35. The nanocarrier of embodiment 34, wherein said nanocarrier has an average particle size ranging from about 10 nm to about 1000 nm (or any value between 10 and 1000 nm, e.g., 15 nm, or any range of values between 10 and 1000 nm, e.g., 100-137 nm, in increments of 1.0 nm).
36. A composition comprising (i)(a) one or more amphiphilic copolymers, wherein each amphiphilic copolymer of said composition independently comprises the amphiphilic copolymer of any one of embodiments 1 to 33 or (i)(b) the nanocarrier of embodiment 34 or 35, and (ii) at least one biologically active substance combined with the one or more amphiphilic copolymers or the nanocarrier.
37. The composition of embodiment 36, wherein the at least one biologically active substance comprises a drug, an anti-carcinogenic compound, a nucleic acid, another small molecule, or any combination thereof.
38. The composition of embodiment 36 or 37, wherein the at least one biologically active substance comprises an anti-carcinogenic compound.
39. The composition of any one of embodiments 36 to 38, wherein the at least one biologically active substance comprises paclitaxel, docetaxel, carbazitaxel, ixabepilone, eribulin, topotecan, irinotecan, SN-38, doxorubicin, daunorubicin, idarubicin, epirubicin, etoposide, omacetaxine, or any combination thereof.
40. The composition of any one of embodiments 36 to 39, wherein the at least one biologically active substance comprises paclitaxel.
41. The composition of any one of embodiments 36 to 40, wherein the at least one biologically active substance comprises let-7 mimic. It should be noted that other nucleic acids may be used instead of or in combination with let-7 mimic. Other suitable nucleic acids include, but are not limited to, other miRNA mimics such as miR-34 mimic, anti-miRs, siRNAs, antisense oligonucleotides, and any combination thereof. Consequently, in all of the embodiments (and claims) herein, any other nucleic acid or combination of nucleic acids may be substituted for let-7 mimic.
42. The composition of any one of embodiments 36 to 41, wherein the at least one biologically active substance comprises a combination of paclitaxel and let-7 mimic.
43. The composition of embodiment 42, wherein the combination of paclitaxel and let-7 mimic is present with the amphiphilic copolymer of embodiment 24 with the paclitaxel being associated with hydrophobic portions of the vitamin E units, and the let-7 mimic being associated with cationic portions of the diethylenetriamine groups.
44. The composition of any one of embodiments 36 to 43, wherein said composition comprises (1) a first amphiphilic copolymer of any one of embodiments 1 to 10, 15 to 16, 19 to 21, 25 to 26 and 30 to 31, and (2) a second amphiphilic copolymer of any one of embodiments 11 to 14 and 17 to 33, the at least one biologically active substance being incorporated within or along at least the second amphiphilic copolymer.
45. The composition of embodiment 44, wherein the first amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 10; the second amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 24; and the at least one biologically active substance comprises paclitaxel and let-7 mimic with the paclitaxel being associated with hydrophobic portions of the vitamin E units, and let-7 mimic being associated with cationic portions of the diethylenetriamine groups.
46. The composition of embodiment 44 or 45, wherein the first amphiphilic copolymer and the second amphiphilic copolymer are present at a weight ratio of from about 1:1 to about 16:1 (or any weight ratio between 1:1 and 16:1, e.g., 8:1, or any range of weight ratios between 1:1 and 16:1, e.g., from about 6.5:1 to about 12.8:1, in increments of 0.1).
47. The composition of any one of embodiments 44 to 46, wherein the first amphiphilic copolymer and the second amphiphilic copolymer are present at a weight ratio of about 8:1.
48. The composition of any one of embodiments 44 to 47, further comprising a third amphiphilic copolymer comprising the amphiphilic copolymer of any one of embodiments 11 to 12, 19 to 23, 25 to 27 and 30 to 33, wherein R represents a crosslinking moiety.
49. The composition of embodiment 48, wherein each R represents thioctic acid.
50. The composition of embodiment 48 or 49, wherein the third amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 12.
51. The composition of any one of embodiments 48 to 50, wherein a weight ratio of (i) a combined weight of the first amphiphilic copolymer and the third amphiphilic copolymer to (ii) the second amphiphilic copolymer is from about 1:1 to about 16:1 (or any weight ratio between 1:1 and 16:1, e.g., 8:1, or any range of weight ratios between 1:1 and 16:1, e.g., from about 6.5:1 to about 12.8:1, in increments of 0.1).
52. The composition of any one of embodiments 48 to 51, wherein a weight ratio of (i) a combined weight of the first amphiphilic copolymer and the third amphiphilic copolymer to (ii) the second amphiphilic copolymer is from about 8:1.
53. The composition of any one of embodiments 36 to 43, wherein said composition comprises (1) a first amphiphilic copolymer of any one of embodiments 11 to 12, 19 to 23, 25 to 27 and 30 to 33, wherein R represents a crosslinking moiety, and (2) a second amphiphilic copolymer of any one of embodiments 11 to 14 and 17 to 33, wherein R represents a cationic moiety, the at least one biologically active substance being incorporated within or along at least the second amphiphilic copolymer.
54. The composition of embodiment 53, wherein each crosslinking moiety comprises thioctic acid.
55. The composition of embodiment 53 or 54, wherein each cationic moiety comprises diethylenetriamine.
56. The composition of any one of embodiments 53 to 55, wherein the first amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 12; the second amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 24; and the at least one biologically active substance comprises paclitaxel and let-7 mimic with the paclitaxel being associated with hydrophobic portions of the vitamin E units, and let-7 mimic being associated with cationic portions of the diethylenetriamine groups.
57. The composition of any one of embodiments 53 to 56, wherein the first amphiphilic copolymer and the second amphiphilic copolymer are present at a weight ratio of from about 1:1 to about 16:1 (or any weight ratio between 1:1 and 16:1, e.g., 8:1, or any range of weight ratios between 1:1 and 16:1, e.g., from about 6.5:1 to about 12.8:1, in increments of 0.1).
58. The composition of any one of embodiments 53 to 57, wherein the first amphiphilic copolymer and the second amphiphilic copolymer are present at a weight ratio of about 8:1.
59. The composition of any one of embodiments 36 to 58, further comprising water.
60. The composition of any one of embodiments 36 to 59, further comprising one or more additional components selected from the group consisting of sodium phosphate, sodium chloride, glucose, HEPES, mannitol, and combinations thereof.
61. The composition of any one of embodiments 36 to 60, wherein said composition does not contain any organic solvents or surfactants.
62. The composition of any one of embodiments 36 to 61, wherein each amphiphilic copolymer is present in any amount of up to about 20 mg/mL (or any amount between about 0.001 mg/mL and 20 mg/mL, e.g., 10.2 mg/mL, or any range of amounts between between about 0.001 mg/mL and 20 mg/mL, e.g., from about 0.01 mg/mL to about 8.002 mg/mL, in increments of 0.001). It should be noted that the each amphiphilic copolymer may be present in any amount depending upon a number of factors including, but not limited to, the size of the amphiphilic copolymer(s), the application (e.g., drug delivery), and the biologically active substance(s) used.
63. The composition of any one of embodiments 36 to 62, wherein each biologically active substance is present in any amount of up to about 6.0 mM (or any amount between about 0.001 mg/mL and 6.0 mg/mL, e.g., 4.2 mg/mL, or any range of amounts between between about 0.001 mg/mL and 6.0 mg/mL, e.g., from about 0.01 mg/mL to about 3.458 mg/mL, in increments of 0.001). It should be noted that each biologically active substance may be present in any amount depending upon a number of factors including, but not limited to, the size of the amphiphilic copolymer(s), the application (e.g., drug delivery), and the biologically active substance(s) used.
II. Method of Making Amphiphilic Copolymers, Nanocarriers and Compositions Containing Amphiphilic Copolymers and/or Nanocarriers
The present invention further provides methods for making amphiphilic copolymers, nanocarriers and compositions containing amphiphilic copolymers and/or nanocarriers. Various embodiments of the present invention relating to methods for making amphiphilic copolymers, nanocarriers and compositions containing amphiphilic copolymers and/or nanocarriers are provided below.
Methods of Making Amphiphilic Copolymers, Nanocarriers and Compositions Embodiments:
64. A method of making the amphiphilic copolymer of any one of embodiments 1 to 33, the nanocarrier of embodiment 34 or 35, or the composition of any one of embodiments 36 to 63, said method comprising: covalently bonding three or more vitamin E units to the polyethylene glycol.
65. The method of embodiment 64, wherein the covalently bonding step comprises: reacting a functionalized polyethylene glycol having a terminal amine end group and a terminal carboxyl end group with one or more divalent linkages; and reacting functional groups on the one or more divalent linkages with the three or more vitamin E units.
66. The method of embodiment 65, wherein the covalently bonding step further comprises: reacting one or more crosslinking moieties with either (i) the polyethylene glycol, (ii) the one or more divalent linkages, or both (i) and (ii).
67. The method of any one of embodiments 64 to 66, further comprising: combining the at least one biologically active substance with the amphiphilic copolymer or the nanocarrier.
68. The method of embodiment 67, wherein the at least one biologically active substance comprises paclitaxel, docetaxel, carbazitaxel, ixabepilone, eribulin, topotecan, irinotecan, SN-38, doxorubicin, daunorubicin, idarubicin, epirubicin, etoposide, omacetaxine, or any combination thereof.
69. The method of embodiment 67 or 68, wherein the at least one biologically active substance comprises paclitaxel.
70. The method of any one of embodiments 67 to 69, wherein the at least one biologically active substance comprises let-7 mimic. As noted above, other nucleic acids may be used instead of or in combination with let-7 mimic with other suitable nucleic acids including, but are not limited to, other miRNA mimics such as miR-34 mimic, anti-miRs, siRNAs, antisense oligonucleotides, and any combination thereof. Consequently, in all of the embodiments (and claims) herein, any other nucleic acid or combination of nucleic acids may be substituted for let-7 mimic.
71. The method of any one of embodiments 67 to 70, wherein the at least one biologically active substance comprises a combination of paclitaxel and let-7 mimic.
72. The method of embodiment 71, wherein the combination of paclitaxel and let-7 mimic is combined with the amphiphilic copolymer of embodiment 24 so that the paclitaxel is associated with hydrophobic portions of the vitamin E units, and the let-7 mimic is associated with cationic portions of the diethylenetriamine groups.
73. The method of any one of embodiments 64 to 72, said method comprising forming a composition comprising (1) a first amphiphilic copolymer of any one of embodiments 1 to 10, 15 to 16, 19 to 21, 25 to 26 and 30 to 31, and (2) a second amphiphilic copolymer of any one of embodiments 11 to 14 and 17 to 33, the at least one biologically active substance being incorporated within or along at least the second amphiphilic copolymer.
74. The method of embodiment 73, wherein the first amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 10; the second amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 24; and the at least one biologically active substance comprises paclitaxel and let-7 mimic with the paclitaxel being associated with hydrophobic portions of the vitamin E units, and let-7 mimic being associated with cationic portions of the diethylenetriamine groups.
75. The method of embodiment 73 or 74, wherein the first amphiphilic copolymer and the second amphiphilic copolymer are present at a weight ratio of from about 1:1 to about 16:1 (or any weight ratio between 1:1 and 16:1, e.g., 8:1, or any range of weight ratios between 1:1 and 16:1, e.g., from about 0.5:1 to about 12.8:1, in increments of 0.1).
76. The method of any one of embodiments 73 to 75, wherein the first amphiphilic copolymer and the second amphiphilic copolymer are present at a weight ratio of about 8:1.
77. The method of any one of embodiments 73 to 76, further comprising forming the composition with a third amphiphilic copolymer comprising the amphiphilic copolymer of any one of embodiments 11 to 12, 19 to 23, 25 to 27 and 30 to 33, wherein R represents a crosslinking moiety.
78. The method of embodiment 77, wherein each R represents thioctic acid.
79. The method of embodiment 77 or 78, wherein the third amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 12.
80. The method of any one of embodiments 77 to 79, wherein a weight ratio of (i) a combined weight of the first amphiphilic copolymer and the third amphiphilic copolymer to (ii) the second amphiphilic copolymer is from about 1:1 to about 16:1 (or any weight ratio between 1:1 and 16:1, e.g., 8:1, or any range of weight ratios between 1:1 and 16:1, e.g., from about 6.5:1 to about 12.8:1, in increments of 0.1).
81. The method of any one of embodiments 77 to 80, wherein a weight ratio of (i) a combined weight of the first amphiphilic copolymer and the third amphiphilic copolymer to (ii) the second amphiphilic copolymer is from about 8:1.
82. The method of any one of embodiments 64 to 72, said method comprising forming a composition comprising (1) a first amphiphilic copolymer of any one of embodiments 11 to 12, 19 to 23, 25 to 27 and 30 to 33, wherein R represents a crosslinking moiety, and (2) a second amphiphilic copolymer of any one of embodiments 11 to 14 and 17 to 33, wherein R represents a cationic moiety, the at least one biologically active substance being incorporated within or along at least the second amphiphilic copolymer.
83. The method of embodiment 82, wherein each crosslinking moiety comprises thioctic acid.
84. The method of embodiment 82 or 83, wherein each cationic moiety comprises diethylenetriamine.
85. The method of any one of embodiments 82 to 84, wherein the first amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 12; the second amphiphilic copolymer comprises the amphiphilic copolymer of embodiment 24; and the at least one biologically active substance comprises paclitaxel and let-7 mimic with the paclitaxel being associated with hydrophobic portions of the vitamin E units, and let-7 mimic being associated with cationic portions of the diethylenetriamine groups.
86. The method of any one of embodiments 82 to 85, wherein the first amphiphilic copolymer and the second amphiphilic copolymer are present at a weight ratio of from about 1:1 to about 16:1 (or any weight ratio between 1:1 and 16:1, e.g., 8:1, or any range of weight ratios between 1:1 and 16:1, from about 6.5:1 to about 12.8:1, in increments of 0.1).
87. The method of any one of embodiments 82 to 86, wherein the first amphiphilic copolymer and the second amphiphilic copolymer are present at a weight ratio of about 8:1.
88. The method of any one of embodiments 64 to 87, further comprising: incorporating the at least one biologically active substance and each amphiphilic copolymer or nanocarrier into an aqueous solution.
The present invention is even further directed to methods of using the amphiphilic copolymers, nanocarriers and compositions containing amphiphilic copolymers and/or nanocarriers. Various embodiments of the present invention relating to methods for using amphiphilic copolymers, nanocarriers and compositions containing amphiphilic copolymers and/or nanocarriers are provided below.
Methods of Using Amphiphilic Copolymers, Nanocarriers and Compositions Embodiments:
89. A method of delivering one or more drugs to a patient, said method comprising: administering an effective amount of the amphiphilic copolymer of any one of embodiments 1 to 33, the nanocarrier of embodiment 34 or 35, or the composition of any one of embodiments 36 to 63 to the patient.
The present invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.
Monomethylterminated poly(ethylene glycol) monoamine, HCl salt (CH3O-PEG-NH2.HCl) with number average molecular weight (Mn) of 5000 g/mol and polydispersity index (PDI) 1.04 (GPC) was purchased from Jenkem Technology (Beijing, China) and used without further purification.
Nα-Fmoc-Nε-Boc-L-lysine (Fmoc-Lys(Boc)-OH) (i.e., Nα-fluorenylmethyloxy-carbonyl-Nε-butyloxycarbonyl-L-lysine), and Nα,Nε-Fmoc-L-lysine (Fmoc-Lys(Fmoc)-OH) were purchased from P3Biosystems (Shelbyville, Ky.).
N,N′-Diisopropylcarbodiimide (DIC), N-hydroxysuccinimide (NHS), ethyldiisopro-pylamine (DIEA), piperidine and trifluoroacetic acid (TFA) and triphosgene were obtained from Alfa Aesar (Ward Hill, Mass.).
Vitamin E was purchased from Sigma (St. Louis, Mo.).
1-Hydroxybenzotriazole hydrate (HOBt) and L-aspartic acid beta-benzyl ester were purchased from AK Scientific (Union City, Calif.).
β-Benzyl-L-aspartate N-carboxy anhydride (BLA-NCA) was carried out by the Fuchs-Farthing method using 1 equivalent of beta-benzyl-L-aspartate and 1.2 equivalents of triphosgene in tetrahydrofuran (THF).
Paclitaxel was purchased from LC Laboratories (Woburn, Mass.).
1,10-Dioctadecyl-3,3,30,30-tetramethylindodicarbocyanine perchlorate (DiD) was purchased from Invitrogen (Grand Island, N.Y.).
3,3′-Dioctadecyloxacarbocyanine, perchlorate (DiO) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) were obtained from Biotium (Hayward, Calif.).
All solvents were of ACS grade or higher and were purchased from Alfa Aesar.
Lipofectamine 2000, LysoTraker Deep Red and Oregon green-labeled paclitaxel were purchased from Life Technologies (Grand Island, N.Y.).
Let-7b-5p mimic and the Cy3 labeled-let-7b-5p mimic were purchased from Bioneer (Alameda, Calif.).
In this example of the present invention, a novel copolymer comprised of polyethylene glycol 5000 (PEG5K), Vitamin E (VE) and thioctic acid (TA) with a molar ratio of PEG:VE:TA at 1:4:4 was prepared. The resulting PEG5K-VE4-TA4 copolymer self-assembled into micelles in aqueous solution, which could be readily crosslinked by a catalytic amount of dithiothreitol to form polydisulfide crosslinks within the hydrophobic region of the micelles. The crosslinked PEG5K-VE4-TA4 micelles had an average particle size of 44 nm, a neutral surface charge and a maximum loading for paclitaxel of 4.3 mM, nearly identical to the characteristics of the non-crosslinked micelles. The crosslinking of the PEG5K-VE4-TA4 micelles not only enhanced thermodynamic and kinetic stability, but also deterred the release of paclitaxel from the micelles. The polydisulfide crosslinks were responsive to the intracellular level of glutathione, which caused rapid disassembly of the micelles and accelerated drug release. Intravenous administration of paclitaxel-loaded crosslinked PEG5K-VE4-TA4 micelles yielded approximately 3-fold and 5-fold higher plasma concentration than that of the non-crosslinked counterparts and Taxol®, respectively. In mice bearing human ovarian SKOV-3 tumors, in vivo imaging study revealed that intravenous injection of DiD-labeled crosslinked micelles resulted in 3-fold higher near infrared fluorescence intensity in SKOV-3 tumors than that of the non-crosslinked micelles at 48 h. Moreover, paclitaxel-loaded crosslinked micelles exerted significantly more potent tumor growth inhibition than the non-crosslinked counterparts and Taxol®. The crosslinked PEG5K-VE4-TA4 nanocarrier system was shown to represent a promising platform for the delivery of hydrophobic anticancer agents.
As discussed further below, nanocarriers were synthesized and evaluated. The reduction-responsive nanocarrier system was based on TA-grafted PEG5K-VE4 copolymer (see,
Synthesis of Copolymers
The PEG5K-VE4-TA4 copolymer was synthesized via stepwise peptide condensation reactions from MeO-PEG-NH2.HCl. PEG5K-NH2 (see, component 1 shown in
Fmoc groups were de-protected by the treatment with piperidine in DMF (v/v 1:4, 100 mL). The PEGylated compound were precipitated and washed with cold ether (200 mL). One more coupling of Fmoc-Lys(Boc)-OH and two more coupling of Fmoc-Lys(Fmoc)-OH were carried out step by step to furnish PEGylated compound 5 shown in
The Boc groups of PEGylated compound 6 were removed by TFA in dichloromethane (v/v 1:3, 20 mL) for 30 min. The mixture was precipitated in cold ether and collected following centrifugation. Two coupling of Fmoc-Lys(Fmoc)-OH and one coupling of thioctic acid to the side amino groups of the PEGylated compound were further carried out according to the typical conjugation procedure. The PEG5K-VE4-TA4 copolymer was recovered from the mixture by repeated precipitation from DMF into diethyl ether. Finally, the copolymer was further purified by dialysis against deionized water for 2 days and lyophilized to obtain an off-white solid. 1H NMR spectra of the copolymers were collected at 20° C. on a 600 MHz Agilent Inova Spectrometer (Santa Clara, Calif.) using CDCl3 as a solvent.
In summary, the reaction scheme shown in
(a) Fmoc-Lys(Bos)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(b)(i) 20% Piperidine in DMF, r.t, 2 h.; and (ii) Fmoc-Lys(Bos)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(c)(i) 20% Piperidine in DMF, r.t, 2 h.; and (ii) Fmoc-Lys(Fmoc)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(d)(i) 20% Piperidine in DMF, r.t, 2 h.; and (ii) Fmoc-Lys(Fmoc)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(e)(i) 20% Piperidine in DMF, r.t, 2 h.; and (ii) α-tocopheryloxyacetic acid, DIC, NHS, DIEA, DMF, r.t, overnight.
(f)(i) 50% TFA in CHCl3, r.t, 1 h.; and (ii) Fmoc-Lys(Fmoc)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(g)(i) 20% Piperidine in DMF, r.t, 2 h.; and (ii) lipoic acid, DIC, NHS, DMF, DMF, r.t, overnight.
Preparation of the PEG5K-VE4-TA4 Micelles
Preparation of the Blank Micelles
The blank micelles were prepared by dissolving 90 mg PEG5K-VE4-TA4 copolymer in 1 mL phosphate buffered saline (PBS) following 20 min sonication. The micelle solution was divided equally into two portions, one of which remained unmodified to be the blank non-crosslinked micelles (B-NCMs). To the second portion, nitrogen gas (N2) was bubbled for 10 min, and 10 μL of DTT (30 mg/mL in PBS) was then added to the micelle solution under a N2 atmosphere. The solution was shaken at 4° C. for 24 h to form the blank crosslinked micelles (B-CMs). The UV absorbance of the micelle solution was measured at 330 nm to confirm the formation of the crosslinks among the TA moieties.
Preparation of Paclitaxel-Loaded Micelles and the NIR Dye-Loaded Micelles
A solvent evaporation method was employed to prepare the drug-loaded micelles similar to the procedure reported in Katragadda U, Fan W, Wang Y, Teng Q, Tan C. “Combined delivery of paclitaxel and tanespimycin via micellar nanocarriers: pharmacokinetics, efficacy and metabolomic analysis”, PLoS One 2013; 8:e58619, the subject matter of which is hereby incorporated by reference in its entirety. Briefly, the varying amount of paclitaxel and 90 mg PEG5K-VE4-TA4 were dissolved in 2 mL chloroform in a 25-mL round-bottom flask, which was rotor evaporated to dryness at room temperature to form a homogenous thin drug-copolymer film. The resulting thin film was further dried overnight under vacuum to remove any residual solvent. Paclitaxel-loaded non-crosslinked micelles (P-NCMs) were formed by hydrating the film in 1 mL PBS followed by 20 min sonication. To prepare the crosslinked micelles, N2 was bubbled into 500 μL P-NCMs for 10 min, and 10 μL DTT (30 mg/mL in PBS) was then added to the micelle solution under a N2 atmosphere. The solution was shaken at 4° C. for 24 h to form paclitaxel-loaded crosslinked micelles (P-CMs).
The loading concentration of paclitaxel in the micelles, defined as the amount of the drugs in the resulting micellar solution per unit volume of PBS, was quantified by HPLC as described below. The encapsulation efficiency of paclitaxel was calculated as the percentage of the incorporated vs. the input paclitaxel.
DiD and paclitaxel were co-loaded into the non-crosslinked (DP-NCMs) and crosslinked micelles (DP-CMs) using the same method as described above with the final DiD concentration as 0.1 mg/mL. DiO and DiI, a lipophilic florescence energy transfer (FRET) pair, were co-loaded into the crosslinked micelles using the same method with a final concentration of 0.25 mg/mL for each dye. All micelle formulations were filtered with 0.22 μM syringe filter for sterilization.
Characterization of the PEG5K-VE4-TA4 Micelles
Size and Zeta Potential
The hydrodynamic diameter and zeta potential of the micelles were recorded by dynamic light scattering (DLS) using a Nano Zetasizer ZS (Malvern Instruments, UK) equipped with He—Ne laser (4 mW, 633 nm) light source and 90° angle scattered light collection configuration.
Transmission Electron Microscopy (TEM)
The morphology of the micelles was observed at room temperature using a Tecnai G2 Spirit TEM (FEI, Hillsboro, Oreg.). A drop of the micellar solution was deposited on a carbon-coated copper grid, dried at room temperature and negatively stained with 1% phosphotungstic acid before TEM visualization.
Critical Micelle Concentration (CMC)
The CMC was determined by fluorescence spectra using pyrene as a fluorescent probe. Pyrene solution (20 μL, 0.5 mM in ethanol) was added into the glass tubes, and the solvent was evaporated at room temperature. B-NCMs or B-CMs solution was diluted in deionized water to the copolymer concentration ranging from 0.1-50 mg/L, which was added to the pyrene-containing glass tubes and gently shaken for 24 h to reach equilibrium before measurement. The fluorescence spectra of pyrene were recorded by a fluorescence spectrophotometer (Perkin Elmer, USA). The excitation wavelength at 331 nm and the emission fluorescence intensity at 373 nm and 393 nm values were used. The CMC was estimated from the threshold of the copolymer concentration, where the intensity ratio 1393/1373 begins to increase markedly.
Stability Studies of the PEG5K-VE4-TA4 Micelles
The storage stability of the PEG5K-VE4-TA4 micelles in PBS was assessed by monitoring paclitaxel concentration and the micelle size following incubation at 4° C. for 4 weeks. To study the stability of the micelles in sodium dodecyl sulfate (SDS), P-NCMs or P-CMs were incubated in PBS containing 2.5 mg/mL SDS at room temperature for 90 min. To evaluate the stability of the micelles in serum, the micelles were incubated in PBS containing 50% fetal bovine serum (FBS) at 37° C. for 24 h. The stability of P-CMs in PBS containing 2 μM or 10 mM GSH was studied at 37° C. for 12 h. The size of the micelles was monitored by DLS. All samples were in triplicates.
In Vitro Release Study of Paclitaxel from the PEG5K-VE4-TA4 Micelles
The release of paclitaxel from the PEG5K-VE4-TA4 micelles was studied by a dialysis method. Paclitaxel-loaded micelles in PBS was inserted into a Slid-A-Lyzer dialysis cassette (Thermo Scientific, Rockford, Ill.) with a 20 KDa MWCO. The cassette was submerged into 50 mL of PBS as the dialysis media at 37° C. and swirled at 100 rpm. At predetermined time intervals, the sample was withdrawn from the cassette, the paclitaxel concentration was quantified by HPLC, and the dialysis media was refreshed to maintain the sink condition. To further investigate the release profile of the crosslinked micelles in response to reduction, the cassettes were also dialyzed against 50 mL PBS containing 2 μM or 10 mM GSH to mimic the extracellular and intracellular GSH level, respectively.
Cell Culture
Human ovarian cancer SKOV-3 cells (American Type Culture Collection, Manassas, Va.) were grown in DMEM medium (Invitrogen, Carlsbad, Calif.) with 2 mM L-glutamine, which was supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin G and 10 μg/mL streptomycin. The cells were maintained at 37° C. with 5% CO2 in a humidified incubator.
Cellular Uptake of the Crosslinked PEG5K-VE4-TA4 Micelles
To study the uptake of the crosslinked micelles, SKOV-3 cells were treated with DiI/DiO-loaded crosslinked micelles for 4, 6, 12 and 24 h. After removing the culture medium, cells were washed 3 times with cold PBS, and then trypsinized and re-suspended in PBS before being subjected to the flow cytometry analysis. Cell-associated fluorescence was analyzed using a BD Accuri C6 Flow Cytometer System (San Jose, Calif.). The fluorescence signals were acquired at the excitation wavelength of 488 nm. The spectral filters of 530±15 nm and 585±20 nm was used to detect DiO and DiI, respectively. All intensities were obtained with the same gain and offset. Data were analyzed using the FlowJo 9.3.1 software (Tree Star, Inc., Ashland, Oreg.) and expressed as the geometric mean of the entire cell population.
Cytotoxicity Assay
SKOV-3 cells were seeded at a density of 6,000 cells/well in 96-well plate, and treated in triplicates with various concentrations of blank micelles (10-3-10 mg/mL) or paclitaxel (1-100 nM) either in the free form, non-crosslink or cross-linked micelles for 72 h. Cells were then fixed with 1% glutaraldehyde, stained with 0.1% crystal violet, and dissolved in 10% acetic acid. The absorbance was quantified at 595 nm on FLUOstar Omega plate reader (Cary, N.C.). The relative cell viability was calculated as the percentage of absorbance of the treated vs. the untreated wells. Results are reported as the means for each triplicate samples.
Tumor Xenograft Murine Model
All animal procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at Mercer University (Approval Number: A1105007). SKOV-3 cells (2×106 in 0.1 mL matrigel/DMEM mixture) were subcutaneously implanted in each flank of 5-6 week-old female athymic nude mice (nu/nu, Charles River, Wilmington.)
Pharmacokinetic Study
When the tumor volumes reached 100-300 mm3, the mice were randomized into three treatment groups (n=3). Each group was i.v. administrated with Taxol®, P-NCMs and P-CMs at an identical dose of 5 mg/kg paclitaxel. The loading concentration of paclitaxel in both P-NCMs and P-CMs was 0.5 mM. At pre-determined time point, namely 5, 15, 30, 45, 60 and 90 min post administration, the blood samples were collected via retro-orbital bleeding. The concentration of paclitaxel in plasma was quantified by HPLC as described below. Non-compartmental analysis was performed to obtain the pharmacokinetic parameters including the elimination half-life (t1/2,e), the total clearance (CLT), and the apparent volume of distribution (Vd,ss) using the WinNonlin software (version 5.1, Pharsight, Sunnyvale, Calif.).
HPLC Methodology
The Waters HPLC system (Milford, Mass.) consisted of a 2795 pump with an autosampler, a 996 photodiode array detector and a Phenomenex (Torrance, Calif.) C8 column (5 μm, 4.6 mm×150 mm). α-Naphthoflavone was used as an internal standard. The detection wavelengths for paclitaxel and α-naphthoflavone were 227 nm and 281 nm, respectively. An isocratic mobile phase of 28% (v/v) sodium phosphate buffer (25 mM, pH 3.0) with 10 mM triethylamine and 72% (v/v) methanol was used at a flow rate of 1.5 mL/min. The plasma samples were extracted by ethyl acetate, reduced to dryness under vacuum, and reconstituted with the HPLC mobile phase prior to HPLC analysis.
In Vivo NIR Imaging and Tissue Distribution
DiD, a lipophilic NIR dye (excitation: 644 nm, emission: 665 nm), was used to label paclitaxel-loaded PEG5K-VE4-TA4 micelles. DiD and paclitaxel were co-loaded into the micelles to form DP-NCMs and DP-CMs with a final paclitaxel concentration of 0.5 mM as described in Section 1.3.2. When the tumor volume reached 100-300 mm3, the mice were randomized into two treatment groups (n=3), and were i.v. administrated with DP-NCMs or DP-CMs at a dose of 1.6 mg/kg DiD. At 0.083, 4, 8, 24 and 48 h post administration, the mice were anaesthetized by isoflurane and the whole-body images of the mice were scanned by Odyssey imaging system (LI-COR, Lincoln, Nebr.). At 24 and 48 h, the mice were euthanized and the major organs including heart, liver, spleen, lung and kidney as well as the tumor tissues were excised and rinsed with PBS. The NIR signals were scanned and analyzed by Odyssey imaging system. The NIR intensity was quantified using the Odyssey Application Software (Version 3.021).
Antitumor Efficacy Study
When the tumor volumes reached 50-100 mm3, the mice were randomized into five treatment groups (n=5): (A) the untreated group; (B) the B-CMs group; (C) the Taxol® group; (D) the P-NCMs group; and (E) the P-CMs group. For Group B, the mice were i.v. administrated with an equal amount of the PEG5K-VE4-TA4 copolymer as Groups D and E. For Groups C, D, and E, the mice were i.v. administered with different paclitaxel formulations at 5 mg/kg twice-weekly for 3 weeks. The final concentration of paclitaxel in P-NCMs and P-CMs was 0.5 mM. The tumor size was measured using a caliper twice-weekly, and the tumor volume was calculated as ½×length×width2. To monitor the overall toxicity, the body weight of all mice from the different groups was measured twice-weekly. The tumors were excised and divided for immediate formalin fixation and flash freezing (−80° C.) for the histology/immunohistochemistry analysis and Western blotting.
Histology and Immunohistochemistry
Formalin-fixed and paraffin-embedded tumor specimens of 3- to 4-μm thickness were sectioned and stained with hematoxylin and eosin (H&E) and the proliferating cell nuclear antigen (PCNA) antibody following the standard protocols.
Western Blot Analysis
Tumor tissue samples (approximately 20-25 mg per sample) were homogenized and lysed using the RIPA buffer supplemented with the protease and phosphatase inhibitors. Tumor lysates containing 35 mg of total proteins were resolved onto 10% SDS-PAGE gels, and transferred onto the nitrocellulose membranes. Blots were probed with the antibodies for PCNA, phosphorylated Akt (p-Akt) and total Akt, all of which were from Cell Signaling (Danvers, Mass.). Anti-β-actin antibody (Santa Cruz Biotechnology, Inc. (Dallas, Tex.) was used as a loading control.
Statistical Analysis
All data are presented as the mean±standard deviation (SD). Data from the different groups were compared using the Student's t-test. A p-value of less than 0.05 was considered statistically significant.
Results and Discussion
Synthesis and Characterization of PEG5K-VE4-TA4 Copolymer
The synthesis of PEG5K-VE4-TA4 is shown in
As shown in
Preparation and Characterization of the PEG5K-VE4-TA4 Micelles
The crosslinking of the micelles was achieved by introducing DTT to the TA units in the micelles with a molar ratio of DTT:TA at 1:10 for 24 h. The UV spectra of the blank PEG5K-VE4-TA4 micelles in water showed that the absorbance of the disulfide-containing thioctic ring at 330 nm, which completely disappeared after crosslinking (see,
The average diameters of the crosslinked micelles (P-CMs) and their non-crosslinked counterparts (P-NCMs) were 43.9±2.6 nm and 45.4±2.5 nm, respectively. Their nearly identical sizes indicate that the cross-linkages among the TA moieties occur within the micelles, which has little influence on the hydrodynamic diameter of the micelles. The polydispersity indexes (PDI) of both formulations were less than 0.15, pointing to the narrow size distribution of the micelles and minimal inter-micellar aggregation during the crosslinking process. As expected, both micelle types had a close-to-neutral surface potential because of the uncharged PEG5K-VE4-TA4 copolymer. The spherical morphology and the size distribution of P-CMs and P-NCMs were confirmed by TEM (see,
To investigate the encapsulation capacity of the micelles for paclitaxel, PEG5K-VE4-TA4 copolymer at 10 mg/mL was used to encapsulate increasing input concentrations of paclitaxel, and the loading concentration and size distribution of the micelles were monitored. As shown in Table 1 and
The CMC of the crosslinked PEG5K-VE4-TA4 micelles was 0.192 mg/L (
In Vitro Stability of PEG5K-VE4-TA4 Micelles
The storage stability of P-NCMs and P-CMs in PBS was evaluated by monitoring the size and paclitaxel concentration. Both micelle types displayed excellent stability in PBS at 4° C. for at least 4 weeks, reflected by little alteration in the particle size and less than 10% decrease in paclitaxel concentration in the micelle solution (see,
When the micelles are administered intravenously, they first encounter the plasma proteins and the blood cells before reaching the target tissues, which may cause the destabilization of micelles and premature release of the payload. It is therefore desirable for the drug-loaded micelles to sustain the destabilizing environment in the bloodstream and remain associated during the circulation. To evaluate the stability of the micelles in serum, the particle size of micelles in 50% serum was monitored. As shown in
It is known that the intracellular GSH level (1-10 mM) is much higher than the extracellular level (2-10 μM). To study the reduction sensitivity of the crosslinked PEG5K-VE4-TA4 micelles, the change in the micelle size triggered by GSH at different time intervals was monitored (see,
In Vitro Release Study
To evaluate the potential of the PEG5K-VE4-TA4 micelles as drug carriers, the release kinetics of paclitaxel from the crosslinked PEG5K-VE4-TA4 micelles were studied in comparison to the non-crosslinked counterparts. As shown in
To study the release of the drug-loaded crosslinked micelles in response to reduction, the release study of P-CMs in PBS with the addition of 2 μM or 10 mM GSH, which corresponds to the extracellular and intracellular GSH levels, respectively, was studied. As shown in
Intracellular Uptake of the Cross/inked PEG5K-VE4-TA4 Micelles
A fluorescence resonance energy transfer (FRET) pair of hydrophobic dyes, DiO (Ex/Em 488/501 nm) and DiI (Ex/Em 501/565 nm), was used to investigate the uptake of the crosslinked PEG5K-VE4-TA4 micelles into the tumor cells. FRET occurs when a donor dye (DiO) and an acceptor dye (DiI) are present within the range of Förster distance, the emission fluorescence from the excited donor dye is used as the excitation energy for the acceptor dye, resulting in the emission of the acceptor fluorescence. Since both DiO and DiI are encapsulated in the micellar core within Förster distance, when the micelles are exited at 488 nm, the emission energy from DiO can be transferred to adjacent DiI, which subsequently gets excited and emits the fluorescence at 565 nm. In contrast, when the micelles disintegrate or the dye molecules are released from the micelles, no fluorescence from DiI is emitted due to the lack of excitation energy. This feature could therefore be exploited to monitor the intracellular uptake of the intact micelles along with the entrapped payload.
The intracellular uptake of the crosslinked PEG5K-VE4-TA4 micelles was studied by directly determining the fluorescence signal of DiO/DiI-loaded micelles in human ovarian cancer SKOV-3 cells. As shown in
Cytotoxicity of the Blank and Paclitaxel-Loaded PEG5K-VE4-TA4 Micelles
To serve as a safe drug carrier system, it is desirable that the empty micelles are inert and devoid of cytotoxicity. Since all of the building blocks of the PEG5K-VE4-TA4 copolymer are nontoxic in nature, the copolymer itself is expected to cause minimal cytotoxicity. To this end, the cytotoxicity of the blank non-crosslinked (B-NCMs) and the blank crosslinked micelles (B-CMs) against SKOV-3 cells was examined. There was little cytotoxicity for either micelle type even at a polymer concentration up to 10 mg/mL (see
Subsequently, the cytotoxicity of paclitaxel-loaded non-crosslinked (P-NCMs) and crosslinked micelles (P-CMs) was examined in SKOV-3 cells at paclitaxel concentration ranging from 1-100 nM for 72 h (see,
Pharmacokinetics of Paclitaxel-Loaded PEG5K-VE4-TA4 Micelles in Mice
In the clinic, paclitaxel is solubilized in Cremophor EL-based formulation (Taxol®) prior to intravenous administration. To evaluate the therapeutic utility of the crosslinked PEG5K-VE4-TA4 micelles as a nanocarrier system for the delivery of paclitaxel, the pharmacokinetics of P-CMs, P-NCM and Taxol® at an identical dose of 5 mg/kg paclitaxel was examined. As shown in
The results shown in Table 2 represent the mean±SD (n=3).
Importantly, intravenous administration of P-CMs resulted in approximately 5-fold and 3-fold higher paclitaxel level in plasma than that of Taxol® and P-NCM, respectively. Non-compartmental analysis on these profiles revealed that compared to Taxol®, the incorporation of paclitaxel within the PEG5K-VE4-TA4 micelles reduced the volume of distribution (Vd,) of paclitaxel by 35% for P-NCMs and 76% for P-CMs. Consequently, the total clearance (CLT) of P-NCM and P-CMs was 45% and 80% of Taxol®, respectively. These results clearly indicate that the PEG5K-VE4-TA4 micelles are able to retain paclitaxel within the micellar core and restrict the drug distribution into the extravascular space, whereas the crosslinking of the micelles further improves the drug retention within the micelles and prolong the circulation time of paclitaxel.
In Vivo Biodistribution and Tumor Accumulation of DiD/Paclitaxel-Dual Loaded PEG5K-VE4-TA4 Micelles in Mice
To further investigate the pharmacokinetic characteristics of paclitaxel-loaded PEG5K-VE4-TA4 micelles, DiD, a hydrophobic near infrared (NIR) fluorescent dye, which enables whole-body imaging with deep tissue penetration and low tissue absorption/scattering, was co-loaded with paclitaxel in the non-crosslinked (DP-NCMs) and the crosslinked (DP-CMs) micelles. The real-time in vivo biodistribution of DP-NCMs and DP-CMs by employing NIR imaging over 48 h was monitored. As shown in
Next, the normal organs and tumor tissues were dissected for NIR imaging (see,
Anticancer Efficacy of Paclitaxel-Loaded PEG5K-VE4-TA4 Micelles in Tumor-Bearing Mice
The major toxicities associated with Taxol® therapy include the hypersensitivity reactions induced by Cremophor EL and peripheral neuropathy caused by paclitaxel. It will be clinically beneficial to develop an alternative formulation for paclitaxel that: (1) requires no Cremophor EL as a solubilizer; and (2) is amenable to a more frequent and lower dosing regimen to maximize the therapeutic window of paclitaxel. Encouraged by the promising pharmacokinetic results demonstrating that the encapsulation of paclitaxel within the PEG5K-VE4-TA4 micelles notably elevated the plasma concentration of paclitaxel and the crosslinking of the micelles further prolonged the circulation time of paclitaxel, the anticancer efficacy of PCMs and P-NCMs in the tumor-bearing mice was evaluated. Although Taxol® is found to be efficacious at a weekly dose of 20 mg/kg, the mice were treated with twice-weekly 5 mg/kg paclitaxel to observe possible potentiation of its anticancer efficacy via the micellar formulations. As shown in
To further evaluate the in vivo anti-proliferative effect of P-CMs, the tumor specimens were examined by histological analysis and immunohistochemistry. H&E staining showed that P-CMs-treated tumor tissues had smaller tumor cells with less nuclear division when compared to the rest groups (see,
In summary, synthesized redox-responsive PEG5K-VE4-TA4 copolymers were synthesized. The PEG5K-VE4-TA4 copolymer readily formed micelles in aqueous solution and can be further crosslinked by a catalytic amount of DTT. The crosslinked PEG5K-VE4-TA4 copolymer micelles demonstrated improved thermodynamic and kinetic stability, which significantly augmented the delivery of paclitaxel to the tumor tissue and enhanced its antitumor efficacy a human ovarian cancer murine model. Constructed from all naturally occurring, biodegradable and/or biocompatible components, this novel crosslinked micellar nanocarrier system represents an interesting and promising platform for the delivery of sparsely aqueous soluble anticancer agents.
In this example of the present invention, a novel cationic copolymer comprised of polyethylene glycol 5000 (PEG5K), Vitamin E (VE) and diethylenetriamine (DET) at 1:4:20 molar ratio was synthesized. The resulting PEG5K-VE4-DET20 copolymer assembled into hybrid micelles when mixed with neutral PEG5K-VE4 copolymer at a 1:8 weight ratio, which were investigated as the nanocarriers for combined delivery of paclitaxel and let-7b mimic. It was determined that the PEG5K-VE4-DET20 hybrid micelles could retain paclitaxel for an extended period and burst release the drug in the presence of cathepsin B, demonstrating the biodegradability of the copolymers. At a N/P ratio of 12:1, the PEG5K-VE4-DET20 hybrid micelles formed stable polyplexes with let-7b mimic, which were efficiently taken up by tumor cells and underwent endosomal escape. In non-small cell lung cancer A549 cells that harbor mutant KRAS, paclitaxel and let-7b mimic-dual loaded micelles (M-PTX/let-7b) markedly potentiated the cytotoxicity of paclitaxel, elevated apoptosis and diminished the invasiveness of tumor cells. In mice bearing subcutaneous A549 xenografts, intravenous administration of M-PTX/let-7b retarded tumor growth more potently than Taxol®. The present example demonstrated the promise of PEG5K-VE4-DET20 hybrid micelles for concurrent delivery of hydrophobic drugs and miRNA mimics.
While PEG5K provided the hydrophilic palisades that confer the stealth property by sterically hindering protein adsorption, the aliphatic chain and aromatic ring of VE aggregated into the hydrophobic core that stabilized the micellar structure and greatly increased the aqueous solubility of hydrophobic drugs. At a 1:4 molar ratio, the conjugation of PEG5K with VE via polylysine linkage resulted in an amphiphilic copolymer that could self-assemble into micelles with excellent loading capacity for paclitaxel, an otherwise sparingly water-soluble drug that requires Cremophor EL and ethanol for solubilization. Two polyasparagine chains carrying N-(2-aminoethyl) aminoethyl groups (DET10), which are known to be protonated at physiological pH and form polyplexes with nucleic acids, were grafted onto PEG5K-VE4 via peptide linkage. The resulting PEG5K-VE4-DET20 copolymer readily formed hybrid micelles with the neutral PEG5K-VE4 copolymer that had superior solubilizing and loading capacity for paclitaxel via hydrophobic interactions, and formed stable polyplexes with let-7b mimic via electrostatic forces (See, the reaction scheme shown in
Synthesis of PEG5K-VE4-DET20
PEG5K-VE4-DET20 copolymer was synthesized via stepwise peptide condensation reactions from MeO-PEG-NH2.HCl as depicted in
In summary, the reaction scheme shown in
(a) Fmoc-Lys(Bos)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(b)(i) 20% Piperidine in DMF, r.t, 2 h.
(b)(ii) Fmoc-Lys(Bos)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(c)(i) and (d)(i) 20% Piperidine in DMF, r.t, 2 h.
(c)(ii) and (d)(ii) Fmoc-Lys(Fmoc)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(e)(i) 20% Piperidine in DMF, r.t, 2 h.
(e)(ii) a-tocopheryloxyacetic acid, DIC, NHS, DIEA, DMF, r.t, overnight.
(f)(i) 50% TFA in CHCl3, r.t, 1 h.
(f)(ii) BLA-NCA, DMF, r.t, 48 h. g, 20 equiv DET, DMF, 40° C., 2h.
Synthesis of PEG5K-VE4
The PEG5K-VE4 copolymer was synthesized via stepwise peptide condensation reactions from MeO-PEG-NH2.HCl as shown in the reaction scheme of
In summary, the reaction scheme shown in
(a) Fmoc-Lys(Fmoc)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(b)(i) 20% Piperidine in DMF, r.t, 2 h.
(b)(ii) Fmoc-Lys(Fmoc)-OH, DIC, HOBt, DIEA, DMF, r.t, 6 h.
(c)(i) 20% Piperidine in DMF, r.t, 2 h.
(c)(ii) a-tocopheryloxyacetic acid, DIC, NHS, DIEA, DMF, r.t, overnight.
1H NMR Spectroscopy
1H-NMR spectra of PEG5K-VE4-Asp20, PEG5K-VE4-DET20 and PEG5K-VE4 were collected on a 600 MHz Agilent Inova Spectrometer (Santa Clara, Calif.) at 20° C. using CDCl3 as a solvent as shown in
Fabrication of the Blank and Drug-Loaded Micelles
Preparation of the PEG5K-VE4-DET20 Hybrid Micelles and Paclitaxel-Loaded Micelles (MPTX)
The PEG5K-VE4-DET20 hybrid micelles were prepared by dry film method. Briefly, 90 mg copolymers consisting of 10 mg PEG5K-VE4-DET20 and 80 mg PEG5K-VE4 were dissolved in 2 mL chloroform in a 25-mL round-bottom flask, which was rotary evaporated to dryness at room temperature to form a homogenous thin drug-copolymer film. The resulting thin film was further dried overnight under vacuum to remove any residual solvent. The micelles were generated by hydrating the film with 1 mL phosphate buffered saline (PBS, pH 7.4) followed by 20 min sonication. Similarly, M-PTX were prepared by adding 0.43 mg of paclitaxel to the same amounts of copolymers prior to the dry film formation. The loading concentration of paclitaxel in the micelles, defined as the amount of the drug in the resulting micelle solution per unit volume of PBS, was quantified by high performance liquid chromatography (HPLC). The encapsulation efficiency of paclitaxel was calculated as the percentage of the incorporated versus the input paclitaxel amount.
Preparation of Let-7b Mimic-Loaded Micelles (M-let-7b) and M-PTX/Let-7b
A predetermined amount (50 nmol) of let-7b mimic was added to the blank micelles or MPTX described above. The mixture was gently vortexed and then incubated at room temperature for 30 min. A series of let-7b-loaded micelles at different N/P ratios were prepared by varying the copolymer concentration. The N/P ratio is defined as the molar ratio between the total amine groups in PEG5K-VE4-DET20 and the phosphate group in let-7b mimic. To evaluate the dissociation of let-7b mimic from the micelleplexes, the micelle solution was incubated at 37° C. in the presence of heparin sulfate. The electrophoretic mobility of let-7b mimic was visualized on the Gel Doc™ XR+ system (Bio-Rad, Hercules, Calif.) with ethidium bromide staining after electrophoresis on a 1% (w/v) agarose gel for 10 min at 100 V in TAE buffer (40 mM Tris-HCl, 1% v/v acetic acid, 1 mM EDTA).
Critical Micelle Concentration (CMC) of the PEG5K-VE4-DET20 Hybrid Micelles
The CMC was determined by the fluorescence spectra using pyrene as a fluorescent probe. Pyrene solution (10 μL, 1 mM in ethanol) was added into the glass tubes, and the solvent was evaporated in darkness at room temperature. The micelle solution was diluted in deionized water to the total copolymer concentration ranging from 0.25-200 mg/L. Four mL of each was added to the pyrene-containing glass tubes and gently shaken for 24 h to reach equilibrium before measurement. The fluorescence spectra of pyrene were recorded by a fluorescence spectrophotometer (Perkin Elmer, Waltham, Mass.) at room temperature. The excitation wavelength at 331 nm and the emission fluorescence intensity at 373 nm and 384 nm were monitored. The CMC was estimated as the total copolymer concentration, where the intensity ratio I373/I383 begins to decrease markedly.
Size and Surface Charge of the PEG5K-VE4-DET20 Hybrid Micelles
The size distribution and zeta potential of the blank micelles, M-PTX, M-let-7b and MPTX/let-7b were assessed by dynamic light scattering (DLS) using a Nano Zetasizer ZS (Malvern Instruments, Malvern, UK). The micelle solution was diluted with deionized water to a final copolymer concentration of 0.5 mg/mL before the measurement. Each batch was analyzed in triplicates.
Transmission Electron Microscopy (TEM)
The morphology of the micelles was observed at room temperature using a Tecnai G2 Spirit TEM (FEI, Hillsboro, Oreg.). A drop of the micelle solution was deposited on a carbon-coated copper grid, dried at room temperature and negatively stained with 1% phosphotungstic acid before TEM visualization.
The Stability of the PEG5K-VE4-DET20 Hybrid Micelles
To evaluate the stability of let-7b mimic in serum, free let-7b mimic and M-PTX/let-7b (N/P ratio 12:1) were incubated in 10% (v/v) FBS at 37° C. followed by the incubation with heparin sulfate for 45 min. The level of let-7b mimic was assessed by gel electrophoresis as described above. The stability of the PEG5K-VE4-DET20 hybrid micelles in PBS was measured by monitoring size at 4° C. for 4 weeks and the paclitaxel loading was assessed on HPLC.
In Vitro Release Study of Paclitaxel and Let-7b Mimic from M-PTX/Let-7b
The release of paclitaxel and let-7b mimic from M-PTX/let-7b was studied in PBS (pH 7.4), or acetate buffered saline (pH 5.5) in the absence or presence of cathepsin B (10 units/mL) by using a dialysis method. The release of paclitaxel from Taxol® and free let-7b mimic were also studied as the controls. Briefly, the micelles were inserted into a Slide-A-Lyzer™ dialysis cassette (MWCO 20 kDa, Thermo Scientific, Rockford, Ill.), which was incubated in 50 mL of deionized water as the dialysis media at 37° C. At predetermined time points, the samples were withdrawn from the cassette, and the paclitaxel concentration was quantified by HPLC. The samples of the dialysis media were collected and lyophilized. The released let-7b mimic was quantified by quantitative real-time PCR (qRT-PCR). The dialysis media was refreshed at each time point to maintain the sink condition.
HPLC Methodology
The concentration of paclitaxel was determined by a Waters HPLC system (Milford, Mass.) consisting of a 2795 pump with an autosampler, a 996 photodiode array detector and a Phenomenex (Torrance, Calif.) C18 column (5 μm, 4.6 mm×150 mm). α-Naphthoflavone was used as an internal standard. Paclitaxel and α-naphthoflavone were detected at wavelengths of 227 nm and 281 nm, respectively. An isocratic mobile phase of 72% (v/v) methanol and 28% (v/v) sodium phosphate buffer (25 mM, pH 3.0) with 10 mM triethylamine was used at a flow rate of 1.2 mL/min.
In Vitro Cytotoxicity Assay
Human NSCLC cell line A549 (American Type Culture Collection, Manassas, Va.) were grown in DMEM medium (Life Technologies) with 2 mM L-glutamine, which was supplemented with 10% FBS, 1% penicillin and streptomycin. The cells were maintained at 37° C. with 5% CO2 in a humidified incubator. To study the cytotoxicity of the blank PEG5k-VE4-DET20 micelles, A549 cells were seeded at a density of 5,000-8,000 cells/well in 96-well plates and treated in triplicates with the micelles (total copolymer concentration 10-104 mg/L), and the cell viability was determined after 72 h. Lipofectamine 2000 was used as a control. To evaluate the cytotoxicity of paclitaxel and let-7b mimic, A549 cells were treated in triplicates with varying concentrations of paclitaxel (1-25 nM) either in the free form, M-PTX or M-PTX/let-7b (let-7b mimic 50 nM) for 72 h. Cells were then fixed with 1% glutaraldehyde, stained with 0.1% crystal violet, and dissolved in 10% acetic acid. The absorbance was quantified at 595 nm on FLUOstar Omega plate reader (Cary, N.C.). The relative cell viability was calculated as the percentage of absorbance of the treated versus the untreated wells.
Cellular Uptake and Transfection Efficiency
The uptake of M-PTX/let-7b into tumor cells was visualized by a laser scanning confocal fluorescence microscope coupled with using Live cell Imaging System (Zeiss, Jena, Germany). A549 cells (1×105) were seeded 24 h prior to the treatment. The endosomes/lysosomes in the cells were stained with LysoTracker Deep Red according to the manufacturer's instruction. Cy3-labeled let-7b mimic and Oregon green-labeled paclitaxel-loaded micelles (N/P ratio 12:1) were gently mixed and incubated at 37° C. for 30 min to form micelleplexes, which were subsequently added to the cells. The cultured cells were monitored by for 48 h, and the fluorescence images of cells were captured by the laser scanning confocal fluorescence microscope using identical imaging parameters. The transfection efficiency of the PEG5K-VE4-DET20 micelles was determined at different N/P ratios with Lipofectamine 2000 as the positive control. A549 cells (2×105 cells/well) were seeded in a 6-well plate and transfected with M-let-7b at a final concentration of 50 nM for 24 h. The transfected cells were then harvested, and the total RNA and proteins were extracted using the TRIzol Reagent (Life Technologies) and RIPA buffer, respectively. The let-7b mimic concentration was quantified by qRT-PCR, and KRAS protein was analyzed by Western blotting.
Colony Formation Assay
A549 cells were treated with the blank micelles, M-PTX, M-let-7b and M-PTX/let-7b at a final concentration of let-7b mimic at 50 nM and paclitaxel at 20 nM for 48 h. The treated cells were harvested and re-seeded in triplicates in 12-well plates at a density of 150 cells/well. The cells were incubated for 14 days. The adherent cell colonies were fixed with 1% glutaraldehyde and stained with 0.1% crystal violet, imaged, and counted using an inverted microscope (Olympus, Tokyo, Japan). Experiments were independently repeated three times.
Apoptosis Assay
Apoptosis was assessed by detecting the externalization of phosphatidyl serine using Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, Calif.). Following the treatments as described above, A549 cells were harvested and stained with Annexin V-FITC and propidium iodide (PI). After incubation for 15 min at room temperature, the samples were analyzed using an Accuri C6 Flow Cytometer System (BD Biosciences).
Invasion Assay
Cell invasion was evaluated using a Boyden chamber system with a polycarbonate membrane (8-μm pore size; Corning, Corning, N.Y.), which was coated with matrigel (BD Bioscience). Following the treatments as described above, A549 cells (105 cells suspended in serum-free media) were seeded into the upper chamber insert, and the lower chamber was filled with DMEM with 10% FBS. After 40 h, the cells remaining on the upper membrane were removed with cotton wool, whereas the cells that had invaded through the membrane were stained with 1% glutaraldehyde and 0.1% crystal violet, imaged, and counted using an inverted microscope. Experiments were independently repeated three times.
Antitumor Efficacy Study
All animal procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at Mercer University (Approval Number: A1105007). A549 cells (1×107 in 0.1 mL matrigel/DMEM mixture) were subcutaneously implanted in the flank region of 5-6 weeks old female athymic nude mice (nu/nu, Charles River, Wilmington, Mass.). When the tumor volumes reached 50-100 mm3, the mice were randomized into six treatment groups (n=5) to receive the following treatments through the tail vein injection twice weekly for four weeks: (A) PBS; (B) the blank micelles; (C) Taxol®; (D) M-PTX; (E) M-let-7b; and (F) M-PTX/let-7b. For group B, the mice received identical amount of copolymers as groups D and F. For groups C and D, the mice were administered with paclitaxel at 5 mg/kg. For groups E and F, the mice were administrated with let-7b at 1 mg/kg. The tumor size was measured using a caliper twice-weekly, and the tumor volume was calculated as ½×length×width2. The body weight of all mice was also measured twice weekly. On day 42, the mice were euthanized and the tumors were excised and divided for immediate formalin fixation and flash freezing (−80° C.).
Histology and Immunohistochemistry (IHC)
Formalin-fixed and paraffin-embedded tumor specimens of 3- to 4-μm thickness were sectioned and stained with hematoxylin and eosin (H&E) and IHC staining of the proliferating cell nuclear antigen (PCNA) antibody following the standard protocols.
RNA Extraction, cDNA Synthesis and qRT-PCR
Total RNA in cells was extracted using TRIzol. The PrimeScript RT Reagent Kit (Clontech, Mountain View, Calif.) was used for reverse transcription of cDNA according to the manufacturer's procedures. The reactions were incubated first at 16° C. for 30 min, at 42° C. for 30 min, and were then inactivated at 85° C. for 5 min. The intracellular level of let-7b was examined by qRT-PCR using TaqMan miRNA probes (Applied Biosystems, Foster City, Calif.) and Premix Ex Tae kit (Clontech) employing an ABI 7500 RT-PCR system (Applied Biosystems), and all of the reactions were run in triplicates. The let-7b level of was normalized to that of U6 snRNA (Applied Biosystems) as an internal control, a ubiquitously expressed small nuclear RNA. The PCR reactions were incubated at 95° C. for 5 minutes, followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute. A comparative threshold cycle (ΔCT) method was used to compare each treatment with the internal control, and the values are expressed as 2−ΔΔCT.
Western Blotting Analysis
Cells or tumor tissue samples (approximately 40-60 mg per sample) were homogenized and lysed using the RIPA buffer supplemented with the protease and phosphatase inhibitors. Cell or tumor lysates containing 30 μg of total proteins were resolved onto 12% SDS-PAGE gels, and transferred onto the nitrocellulose membranes. Blots were probed with the antibodies for phospho-ERK1/2 (p-ERK1/2), ERK1/2, cleaved caspase-3, caspase-3, E-cadherin, vimentin, HMGA2, PCNA, phosphor-AKT (p-AKT) and AKT, all of which were from Cell Signaling (Danvers, Mass.). Antibodies for KRAS and β-actin were from Santa Cruz Biotechnology, Inc. (Dallas, Tex.).
Statistical Analysis
All data are presented as the mean±standard deviation (SD). Data from the different groups were compared using the Student's t-test. A p-value of less than 0.05 was considered statistically significant, as indicated by asterisks in the figures.
Results and Discussion
Synthesis of PEG5K-VE4-DET20 and PEG5K-VE4 Copolymers
PEG5K-VE4-DET20 and PEG5K-VE4 were synthesized via stepwise peptide condensation reactions using PEG5K-NH2 as the starting material, as shown in
Formulation Development of the PEG5K-VE4-DET20 Hybrid Micelles
The PEG5K-VE4-DET20 copolymer formed micelles readily in aqueous solution with an average diameter of 88 nm and a zeta potential of 30 mV. See, for example, the data in Table 3 below.
The CMC of the PEG5K-VE4-DET20 micelles was 10.75 mg/L as shown in Table 3 and
Characterization of the PEG5K-VE4-DET20 Micelleplexes
The formation of polyplexes between let-7b mimic and the PEG5K-VE4-DET20 hybrid micelles was studied by a gel retardation assay. As shown in
The size distribution and zeta potential of the blank hybrid micelles, M-PTX and M-PTX/let-7b micelles were shown in
The incorporation of paclitaxel had negligible effect on the size (60 nm) and surface charge (11.2 mV) of the micelles. The polyplexation of let-7b mimic reduced the zeta potential of the micelles to about 7 mV, but minimally influenced the size. The hybrid micelles displayed excellent stability in PBS at 4° C. for at least 4 weeks, reflected by little change in the size and the loading concentration of paclitaxel (
Release of Paclitaxel and Let-7b Mimic from the PEG5K-VE4-DET20 Micelleplexes
To assess the ability of the PEG5K-VE4-DET20 hybrid micelles to function as a nanocarrier system for paclitaxel and let-7b mimic, the release kinetics of the payloads from M-PTX/let-7b was evaluated. As shown in
Cellular Uptake of the PEG5K-VE4-DET20 Micelleplexes
Endosomal escape is essential to achieve efficient transfection of nucleic acids. To track the uptake and intracellular fate of M-PTX/let-7b, confocal laser scanning microscope was employed to visualize Cy3-labeled let-7b mimic, Oregon green-labeled paclitaxel and LysoTracker Deep Red-labeled endosomes/lysosomes. As shown in
Let-7b reconstitution in A549 cells treated with M-let-7b was further evaluated by qRT-PCR and Western blotting. As shown in
Antiproliferative Activity of M-PTX/Let-7b
An important consideration when devising drug carrier systems is the minimal toxicity elicited by the carrier materials. To this end, the cytotoxicity of the blank PEG5K-VE4-DET20 hybrid micelles in A549 cells was examined. It was found that the blank micelles were devoid of cytotoxicity at a total polymer concentration up to 10 g/L (
As a driver oncogene, mutant KRAS constitutively activates MEK/ERK and PI3K/AKT signaling pathways, both of which are crucial to the survival and proliferation of tumor cells. To further investigate the molecular mechanism responsible for the antiproliferative effect of MPTX/let-7b in A549 cells that harbor KRASG12S mutation, the activation of ERK and AKT, two KRAS downstream effectors, was examined. As shown in
Induction of Apoptosis by M-PTX/Let-7b
Apoptosis in A549 cells was evaluated by staining with Annexin V-FITC and propidium iodide (PI). Following 48 h incubation, M-let-7b and M-PTX induced late apoptotic cell population (Annexin V+/PI+) by 4% and 11%, respectively. Strikingly, M-PTX-let-7b drastically increased late apoptotic cells to 27% (
Blockage of Tumor Cell Invasion by M-PTX/Let-7b
Epithelial-mesenchymal transition (EMT) converts adherent epithelial cells to motile mesenchymal cells, which is an important process during tumor invasion and metastasis. To evaluate the impact of M-PTX/let-7b on the invasiveness of tumor cells, the transwell invasion assay was carried out in A549 cells. It was found that the number of invading cells was diminished following the treatment of M-PTX/let-7b, whereas M-let-7b or M-PTX only modestly reduced invasion (
Superior Antitumor Efficacy of M-PTX/Let-7b in NSCLC Tumor-Bearing Mice
To explore the therapeutic application of the PEG5K-VE4-DET20 hybrid micelles as nanocarriers for concurrent delivery of miRNA and chemotherapy in vivo, the antitumor efficacy of M-PTX/let-7b was studied in athymic mice bearing subcutaneous A549 xenografts. As shown in
To further evaluate the therapeutic efficacy of paclitaxel/let-7b combination, the let-7b level in the tumor tissues by qRT-PCR was measured. It was found that, while neither Taxol® nor MPTX altered let-7b expression in the tumor, the treatment with M-let-7b or M-PTX/let-7b augmented the let-7b level by 5-7 fold (p<0.01,
Associated with poor prognosis and drug resistance, KRAS mutations are highly prevalent in human cancers including NSCLC, pancreatic and colorectal cancers. Although molecularly targeted therapies are being developed to directly block mutant KRAS-driven oncogenesis, conventional chemotherapeutic drugs including paclitaxel are still the mainstay for treating KRAS mutant cancers. The present work provides proof-of-concept that let-7 replacement therapy may be exploited to enhance the anticancer efficacy of paclitaxel in KRAS mutant cancers.
In summary, the herein-described self-assembling PEG5K-VE4-DET20 hybrid micelles exhibited excellent transfection efficiency and minimal toxicity with particle size less than 100 nm. Paclitaxel and let-7b mimic dual loaded micelleplexes markedly potentiated the antiproliferative activity of paclitaxel, which was accompanied by increased apoptosis and reverted EMT phenotype in KRAS mutant tumor cells. In mice bearing KRAS mutant tumor xenografts, intravenous administration of M-PTX/let-7b resulted in significantly enhanced antitumor efficacy, demonstrating the PEG5K-VE4-DET20 hybrid micelles as a robust platform for the dual delivery of hydrophobic drugs and miRNA mimics, as well as the therapeutic potential of let-7b mimic as a chemosensitizer for KRAS mutant cancers.
It should be understood that although the above-described amphiphilic copolymers, nanocarriers, compositions and/or methods are described as “comprising” one or more components or steps, the above-described amphiphilic copolymers, nanocarriers, compositions and/or methods may “comprise,” “consists of,” or “consist essentially of” the above-described components, features or steps of the amphiphilic copolymers, nanocarriers, compositions and/or methods. Consequently, where the present invention, or a portion thereof, has been described with an open-ended term such as “comprising,” it should be readily understood that (unless otherwise stated) the description of the present invention, or the portion thereof, should also be interpreted to describe the present invention, or a portion thereof, using the terms “consisting essentially of” or “consisting of” or variations thereof as discussed below.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, an amphiphilic copolymer, a nanocarrier, composition and/or method that “comprises” a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the method and/or computing system.
As used herein, the transitional phrases “consists of” and “consisting of” exclude any element, step, or component not specified. For example, “consists of” or “consisting of” used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of” or “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of” or “consisting of” limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.
As used herein, the transitional phrases “consists essentially of” and “consisting essentially of” are used to define an amphiphilic copolymer, a nanocarrier, composition and/or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.
Further, it should be understood that the herein-described amphiphilic copolymers, nanocarriers, compositions and/or methods may comprise, consist essentially of, or consist of any of the herein-described components, features and steps, as shown in the figures with or without any feature(s) not shown in the figures. In other words, in some embodiments, the amphiphilic copolymers, nanocarriers, compositions and/or methods of the present invention do not have any additional features other than those shown in the figures, and such additional features, not shown in the figures, are specifically excluded from the amphiphilic copolymers, nanocarriers, compositions and/or methods. In other embodiments, the amphiphilic copolymers, nanocarriers, compositions and/or methods of the present invention do have one or more additional features that are not shown in the figures.
While the specification has been described in detail with respect to specific embodiments thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing, may readily conceive of alterations to, variations of, and equivalents to these embodiments. Accordingly, the scope of the present invention should be assessed as that of the appended claims and any equivalents thereto.
This application is being filed as a PCT International Patent Application in the name of The Corporation of Mercer University, a U.S. corporation, claiming priority to U.S. Provisional Patent Application Ser. No. 61/980,786 filed on 17 Apr. 2014 and entitled “VITAMIN E-BASED NANOCARRIERS FOR DRUG DELIVERY AND METHODS OF MAKING AND USING THE SAME.”
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/026535 | 4/17/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61980786 | Apr 2014 | US |
