WO2010025176A2

WO2010025176A2 - Mcp-1 delivery system

Info

Publication number: WO2010025176A2
Application number: PCT/US2009/055027
Authority: WO
Inventors: Gary L. Bowlin; Matthew J. Smith; Kimber L. White, Jr.
Original assignee: Organogenesis, Inc.
Priority date: 2008-08-28
Filing date: 2009-08-26
Publication date: 2010-03-04
Also published as: WO2010025176A3

Abstract

The present invention is directed to methods and compositions related to electroprocessed drug delivery devices. An electroprocessed drug delivery device of the present invention can, for example, be made to comprise an electrospun scaffold (e.g., comprising polydioxanone) incorporated with Monocyte Chemotactic Protein- 1 (MCP-I). The electroprocess drug delivery device can be used to influence macrophage infiltration and adherence, and to allow extended chemokine release.

Description

MCP-I DELIVERY SYSTEM

Cross-Reference to Related Applications

This application claims the benefit of priority to U.S. Provisional Application No. 61/092,544, filed on August 28, 2008, the contents of which are hereby incorporated in their entirety.

Background of Invention

Recently, electrospun fibers have been explored as potential drug delivery/controlled release devices. The first reported use of electrospun fibers in controlled delivery applications was published in 2002 by Kenawy et al., in which the authors explored the release of the antibiotic tetracycline hydrochloride from electrospun poly(ethylene-co-vinylacetate) (PEVA) and poly(lactic acid) (PLA) (Kenawy et al. (2002) 81, 57-64, J. Control Release). Results of that study indicated that electrospun PEVA and 50/50 PEVA/PLA demonstrated prolonged release of tetracycline, obtaining larger percent release levels than other materials tested over five days, the length of the study. In contrast, films of the same materials did not demonstrate continued release, plateauing within 24 hours. Following that published success, others have used electrospun fibers as a vehicle for controlled release of other antibiotics, pain relievers, and proteins. The possibility of using electrospun polymers as controlled release devices suggests potential for improving cell infiltration into bioresorbable scaffolds (with the incorporation of an appropriate chemokine), which could potentially result in improved angiogenesis and scaffold remodeling.

This project included initial investigations into the use of electrospun PDO as a novel chemokine delivery device in order to promote macrophage interaction with and infiltration into purely synthetic polymers. It is a widely held belief in tissue engineering that cells do not readily interact with synthetic materials, other than via the typical formation of a fibrotic capsule around such materials. The driving premise of the design component to this project was to promote macrophage adherence and, more importantly, infiltration into synthetic electrospun scaffolds by incorporating a chemokine into the scaffold during the electrospinning process.

One important premise of the in situ approach to tissue engineering is the idea that the body, when appropriately stimulated, can remodel an implanted tissue engineered device to produce new functional tissues and/or organs. A number of chemokines, including MCP-I and RANTES, have been identified as important to the vascular remodeling process. Of these, MCP-I has received increasing attention in the biomedical community due to its potent chemoattractant effects on mononuclear phagocytes and for its role in tumor vasculogenesis. MCP-I is more potent than many other chemokines, including RANTES, potentially due to its affinity for other CC chemokine receptors (such as the RANTES receptor, CCR5), in addition to its high affinity for its own receptor (CCR2).

Summary A novel chemokine delivery system has been developed, in which electrospun polydioxanone (PDO) scaffolds incorporated with Monocyte Chemotactic Protein- 1 (MCP- 1) were fabricated and examined for their potential to influence macrophage infiltration/adherence, and to allow extended chemokine release. Over the course of 120 hours, MCP-I released into supernatant peaked at 24 hours and was detectable by enzyme- linked immunosorbent assay (ELISA) only when added to PDO solutions at 3000 ng/ml prior to electrospinning. The biological activity of PDO/MCP-1 hybrids was characterized for their influence on macrophage adherence/infiltration of scaffolds. Results demonstrated an increasing dose-responsive trend with increasing MCP-I concentration.

Brief Description of the Figures

Figure 1. Results of test-tube ELISA for macrophage adherence/infiltration to electrospun PDO/MCP-1 hybrid structures. Background (BG) = PDO (without MCP-I) cultured in media without cells; control cultures (PDO with 0 ng/ml MCP-I) cultured with cells. (A) Figure is from a single experiment that is representative of two experiments. Values represent the mean O. D. ± SE at 450 nm from four samples. (B) Percent of control presenting combined results of two experiments. Asterisks indicate statistically significant differences from control cultures; *p < 0.05; **p < 0.01.

Figure 2. Histology of PDO/MCP-1 hybrids following overnight culture with splenic adherent macrophages. Samples are representative of each group from the test tube ELISA study for which scaffolds and macrophages were cultured overnight. (A) pure PDO cultured in media without cells; (B) pure PDO cultured with cells; (C) PDO/MCP-1 hybrid containing 3 ng/ml MCP-I cultured with cells; (D) PDO/MCP-1 hybrid containing 30 ng/ml MCP-I cultured with cells; (E) PDO/MCP-1 hybrid containing 300 ng/ml MCP-I cultured with cells; (F) PDO/MCP-1 hybrid containing 3000 ng/ml MCP-I cultured with cells. Samples were stained with H&E. Magnification = 4OX.

Figure 3. Results of ELISA for MCP-I released from electrospun PDO/MCP-1 hybrid structures. Results are reported as the concentration (in pg/ml) of MCP-I detected, and values represent the mean ± SE derived from four material samples. "LOQ" represents the limit of quantification, identified from the lowest concentration in the linear portion of the standard curve. Asterisks indicate statistically significant differences from control (0 ng/ml); *p < 0.05; **p < 0.01.

Detailed Description of the Invention Example:

Electrospun Polydioxanone Scaffolds Incorporated with Monocyte Chemotactic Protein-1 Preparation

Monocyte Chemotactic Protein-1 (MCP-I; Antigenix America Inc, Huntington Station, NY, USA) was prepared in sterile water with 0.1% bovine serum albumin at 30 μg/ml and stored at -20 ⁰C until use. PDO (2 ml; 100 mg/ml in HFP) was electrospun without MCP-I or blended with MCP-I at four different concentrations (3 ng/ml, 30 ng/ml, 300 ng/ml, or 3000 ng/ml) prior to electrospinning onto a rectangular mandrel (7.5 cm x 1 cm x 0.5 cm). Polymer solutions were loaded into 3-ml Becton-Dickenson syringes fitted with 18-gauge blunt-tipped needles and dispensed at 6 ml/hr using a KD Scientific syringe pump. During electrospinning, a fixed voltage of +22 kV was applied to the needle by a Spellman CZElOOOR high voltage power supply (Spellman High Voltage Electronics Corp., Hauppauge, NY, USA), while a fixed voltage of-10 kV was applied to a target 15 cm behind the grounded mandrel using a second CZElOOOR. The mandrel was allowed to rotate at 500 RPM and translate at 2 cm/s over a distance of ± 3.75 cm during electrospinning in order to ensure a random, even fiber distribution; the air gap distance was maintained at 28 cm during electrospinning.

Evaluation

In order to evaluate both the presence of MCP-I in PDO/MCP-1 electrospun hybrid scaffolds and the biological activity of these structures, electrospun PDO and electrospun PDO/MCP-1 hybrid materials were cultured overnight with splenic adherent macrophages and then evaluated for the presence of macrophages on and/or within the structures using a test tube ELISA procedure. Results of the second test tube ELISA conducted on these materials are shown in Figure IA; the first experiment consisted of fewer test groups, including only Background (BG), 0 ng/ml, and 3000 ng/ml groups. Percent of control values (for this second experiment) for each of the groups were: 102.3% (BG), 91.8% (3 ng/ml), 100% (30 ng/ml), 141.7% (300 ng/ml), and 170.8% (3000 ng/ml). These results demonstrate a dose-responsive increase in macrophage adherence/infiltration with increasing MCP-I concentration, reaching the level of statistical significance at 3000 ng/ml. Background (BG) cultures consisted of PDO cultured in media without cells and was utilized to account for incomplete rinsing/removal of unbound antibody from materials. Experimental control cultures (PDO scaffolds devoid of MCP-I but cultured with cells) were not statistically different from background control cultures. Results for this experiment are given as the O. D. at 450 nm, as there is no standard for this assay. Figure IB presents percent of control combining results from two experiments (the first experiment consisted only of BG, control, and 3000 ng/ml treatment groups). The results presented in Figure IB show a similar dose-responsive increase as seen in Figure IA. Mean percent controls for each group were: 83.7% (BG), 91.9% (3 ng/ml), 100% (30 ng/ml), 141.8% (300 ng/ml), and 275.2% (3000 ng/ml). The percent of control mean for the 3000 ng/ml group was statistically significantly greater than the percent of control for all other treatment groups.

Histological Analysis

Figure 2 depicts representative histology cross-sections of samples from each experimental group from a test tube ELISA study on PDO/MCP-1 hybrids following overnight culture with splenic adherent macrophages (5xlO⁶/ml, 0.75 ml). Samples were stained with hematoxylin and eosin (H&E) and imaged using a Nikon Eclipse TE300 equipped with a Nikon DXM 1200 digital camera at 4OX magnification. Figure 2 A depicts the PDO scaffold following culture without cells, while Figure 2B depicts pure PDO cultured with cells and demonstrates very few cells adhered to scaffolds. The remaining parts of Figure 2 are representative of PDO/MCP-1 hybrids containing varying concentrations of MCP-I as follows: (C) 3 ng/ml, (D) 30 ng/ml, (E) 300 ng/ml, and (F) 3000 ng/ml. There appear to be increasing numbers of cells present within the electrospun structures with increasing MCP-I concentration.

Characterization via ELISA Electrospun PDO scaffolds incorporated with MCP-I were also examined for their potential to produce extended MCP-I release. Over the course of 120 hours, MCP-I released into supernatant peaked at 24 hours and was detectable by ELISA at levels as high as 23.5 pg/ml, but only when MCP-I was added to PDO solutions at 3000 ng/ml prior to electrospinning (Figure 3). At the highest electrospun MCP-I concentration, released MCP-I levels were statistically significantly greater than released MCP-I from all other concentrations at all time points with the exception of the 72 hour measurement. There were no other statistically significant differences between test groups.

Discussion In designing the PDO/MCP-1 electrospun hybrid structures, the aim was to incorporate approximately 100 ng MCP-I per 6-mm diameter circular disc of material at the high dose. Based upon the area of a 6-mm circular disc, the mandrel dimensions (1 cm x 7.5 cm x 0.5 cm) and assuming a uniform thickness of the electrospun material, the amount of MCP-I required per ml of PDO was calculated to be 3000 ng. Accordingly, PDO/MCP-1 hybrids were fabricated containing MCP-I at 3 ng/ml, 30 ng/ml, 300 ng/ml, and 3000 ng/ml in order to obtain theoretical MCP-I concentrations of 0.1 ng/disc, 1.0 ng/disc, 10.0 ng/disc, and 100 ng/disc.

Before proceeding further, it was important to examine whether or not MCP-I was indeed present in the electrospun structures, given that the chemokine was added to PDO/HFP solutions prior to electrospinning. The use of a test tube ELISA after overnight culture of materials with macrophages allowed not only for a determination of the presence or absence of MCP-I but also an indication of the biological activity of any MCP-I present. Results of the test tube ELISA demonstrated a dose-responsive increase in macrophage adherence/infiltration to scaffolds with increasing MCP-I concentration, indicating that indeed, MCP-I was present in the structures and that it was biologically active.

Histological evaluations of samples from each group in this experiment were completed in order to assess cell infiltration. Histology micrographs of representative samples of PDO/MCP-1 hybrids following overnight culture with macrophages in test tubes were consistent with results from the test tube ELISA. Specifically, the histology images demonstrated increasing numbers of adherent macrophages that infiltrated through the thickness of the scaffolds with increasing MCP-I concentration.

It was also important to consider the release profile of MCP-I from electrospun PDO structures. It has been reported that MCP-I expression is increased following arterial wall injury, however the upregulation was transient, with levels returning to baseline after 3 to 4 days (Schober and Zernecke (2007) 97, 730-737, Thromb. Haemost). It may therefore be prudent to extend the MCP-I levels in order to allow for a greater cellular response, increased infiltration, and potentially increased vascular remodeling and angiogenesis. The time-dependent MCP-I released from electrospun PDO was detectable only for the highest concentration of incorporated MCP-I and appeared to peak at 24 hours in culture.

From the experimental design for these hybrid structures and with some reasonable assumptions, we can estimate the percentage of MCP-I released over the 120-hour assay period. It is reasonable to assume 10% loss prior to and during electrospinning as a result of MCP-I left behind in the scintillation vial and/or syringe and as a result of fibers not collected on the rectangular portion of the mandrel. To insure that the estimated MCP-I in the structures is not over-estimated, a loss of 30 % of the remaining MCP-I is assumed to occur during the disinfection and purge, leaving approximately 63% of the predicted MCP- 1 concentration remaining in each disc. For the 100 ng/disc level, this equates to 63 ng MCP-I per disc. A mean release at 24 hours of 23.54 pg/ml in a 200 μl sample equates to 2.354 pg of MCP-I released from the disc, or 0.007%.

It is to be appreciated that combinations of additional/alternative natural and/or synthetic biomaterials can be used in accordance with the subject invention. In one aspect, a blended structure comprising polycaprolactone (PCL) and type I collagen can be created and subsequently implemented as a chemokine delivery system. More particularly, for example, the PCL/type I collagen structure can be loaded with MCP-I, such that this vascular prosthetic can be capable of promoting functional in situ development of an artery segment.

Claims

We Claim:

1. A method for producing an electrospun drug delivery device, comprising

blending at least one synthetic polymer and a cytokine to form a solution; and producing a fibrous electrospun scaffold by electrodepositing the solution upon a rotating mandrel.

2. The method of claim 1, wherein the cytokine is a chemokine.

3. The method of claim 1, wherein the chemokine is Monocyte Chemotactic Protein- 1 (MCP-I).

4. The method of claim 3, wherein the concentration of MCP-I is greater than about 3 ng/ml.

5. The method of claim 3, wherein the concentration of MCP-I is greater than about 3000 ng/ml.

6. The method of claim 1, further comprising enhancing monocyte adherence within the scaffold via increasing MCP-I concentration within the device.

7. The method of claim 1 , wherein the at least one synthetic polymer is a poly (ether) ester.

8. The method of claim 7, wherein the poly (ether) ester is polydioxanone (PDO).

9. A method for inducing angiogenesis, comprising: producing a fibrous electrospun scaffold comprising a synthetic polymer and a chemokine via electroprocessing, wherein the amount of chemokine is sufficient to promote monocyte infiltration within the scaffold.

10. The method of claim 9, wherein the chemokine is Monocyte Chemotactic Protein- 1 (MCP-I).

11. The method of claim 10, wherein the concentration of MCP-I is greater than about 3 ng/ml.

12. The method of claim 10, wherein the concentration of MCP-I is greater than about 3000 ng/ml.

13. The method of claim 9, wherein the synthetic polymer is a poly (ether) ester.

14. The method of claim 7, wherein the poly (ether) ester is polydioxanone (PDO).

15. An electrospun engineered matrix, comprising: at least one natural polymer; at least one synthetic polymer; and an angiogenic-inducing factor.

16. The matrix of claim 15, wherein the at least one synthetic polymer is selected from the group consisting of poly (ether) ester is polydioxanone (PDO) and polycaprolactone (PCL).

17. The matrix of claim 15, wherein the at least one natural polymer is type I collagen.

18. The matrix of claim 15, wherein the angiogenic-inducing factor is Monocyte

Chemotactic Protein (MCP-I).

19. The matrix of claim 18, wherein the concentration of MCP-I is greater than about 3 ng/ml.

20. The matrix of claim 18, wherein the concentration of MCP-I is greater than about 3000 ng/ml.