Antimicrobial reverse thermal gel and methods of forming and using same

Abstract

Antimicrobial polymers, composition including the polymers, and methods of forming and using the polymers and compositions are disclosed. The compositions have reverse thermal gelling properties and transform from a liquid to a gele.g., near or below body temperature, such as a skin temperature in an operating room. The polymers and compositions can be applied to a surface, such as a surface of skin proximate an incision site prior to surgery, to provide a relatively sterile area near the site.

Claims

1. An antimicrobial reverse thermal gel composition comprising: a polymer having an antimicrobial functional group, wherein the antimicrobial reverse thermal gel composition is liquid at a temperature of about 20 C. or less and the antimicrobial reverse thermal gel material transitions to a gel at a temperature of about 25 C. or more, and wherein the polymer comprises a polyurea-polyurethane polymer backbone.

2. The antimicrobial reverse thermal gel composition of claim 1, wherein the polymer comprises a copolymer comprising a reverse thermal gel block.

3. The antimicrobial reverse thermal gel composition of claim 1, wherein the polymer comprises an antimicrobial functional group block.

4. An antimicrobial reverse thermal gel composition comprising: a polymer having an antimicrobial functional group, wherein the antimicrobial reverse thermal gel composition is liquid at a temperature of about 20 C. or less and the antimicrobial reverse thermal gel material transitions to a gel at a temperature of about 25 C. or more, wherein the polymer comprises a general formula of: ##STR00005##

5. The antimicrobial reverse thermal gel composition of claim 4, where x ranges from about 17 to about 350.

6. The antimicrobial reverse thermal gel composition of claim 4, where y ranges from about 18 to about 61.

7. The antimicrobial reverse thermal gel composition of claim 4, where m ranges from about 6 to about 22.

8. The antimicrobial reverse thermal gel composition of claim 4, where n ranges from about 15 to about 900.

9. The antimicrobial reverse thermal gel composition of claim 4, wherein a percent of PNIPAAm conjugation ranges from about 10 percent to about 50 percent.

10. The antimicrobial reverse thermal gel composition of claim 4, wherein where x ranges from about 17 to about 330, y ranges from about 18 to about 61, and m ranges from about 6 to about 22.

11. The antimicrobial reverse thermal gel composition of claim 1 comprising a general formula of: ##STR00006## where x ranges from about 17 to about 350; y ranges from about 18 to about 61; n ranges from about 15 to about 900; and m ranges from about 6 to about 22.

12. The antimicrobial reverse thermal gel composition of claim 11, wherein a percent of PNIPAAm conjugation ranges from about 10 percent to about 50 percent.

13. The antimicrobial reverse thermal gel composition of claim 1, wherein the gelling time of the composition at a temperature of about 30 C. to about 45 C. ranges from about 5 to about 30 seconds.

14. A composition comprising the antimicrobial reverse thermal gel composition of claim 1.

15. The composition of claim 14, further comprising a diluent.

16. The composition of claim 15, wherein the diluent comprises water.

17. The composition of claim 15, wherein the diluent comprises alcohol.

18. The antimicrobial reverse thermal gel composition of claim 11, where x ranges from about 17 to about 330; y ranges from about 18 to about 51; n ranges from about 15 to about 800; and m ranges from about 6 to about 20.

Description

BRIEF DESCRIPTION OF THE DRAWING FIGURES

(1) A more complete understanding of exemplary embodiments of the present disclosure may be derived by referring to the detailed description and claims when considered in connection with the following illustrative figures.

(2) FIG. 1 illustrates epidermal cell detachment during removal of surgical incision drape.

(3) FIG. 2 illustrates an exemplary polymer formulation in accordance with various embodiments of the disclosure.

(4) FIGS. 3 (A) and (B) illustrate gelling properties of a composition in accordance with exemplary embodiments of the disclosure.

(5) FIG. 4 illustrates antibacterial activity of an exemplary composition in accordance with exemplary embodiments of the disclosure.

(6) FIG. 5 illustrates bacterial growth after inoculation with used surgical incision drapes.

(7) FIG. 6 illustrated another exemplary polymer formulation in accordance with various embodiments of the disclosure.

(8) FIG. 7 illustrates effects of exemplary compositions on cultures of Staphylococcus aureus subsp. aureus Mu3 (MRSA/hetero-VISA) in accordance with various embodiments of the disclosure.

(9) FIG. 8 illustrates effects of exemplary compositions on cultures of Staphylococcus epidermidis in accordance with various embodiments of the disclosure.

(10) FIG. 9 illustrates effects of exemplary compositions on cultures of Staphylococcus aureus subsp. aureus Mu3 (MRSA/hetero-VISA) in accordance with additional embodiments of the disclosure.

(11) FIG. 10 illustrates effects of exemplary compositions on cultures of Staphylococcus aureus in accordance with further embodiments of the disclosure.

(12) FIG. 11 illustrates effects of exemplary compositions on stationary-phase cultures of E. coli. in accordance with further embodiments of the disclosure.

(13) FIG. 12 illustrates absorbance percent of exemplary compositions in accordance with further embodiments of the disclosure.

(14) It will be appreciated that the figures are not necessarily drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help to improve understanding of illustrated embodiments of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE DISCLOSURE

(15) The description of exemplary embodiments of the present disclosure provided below is merely exemplary and is intended for purposes of illustration only; the following description is not intended to limit the scope of the invention disclosed herein. Moreover, recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features or other embodiments incorporating different combinations of the stated features.

(16) Various exemplary embodiments of the disclosure provide a reverse thermal gel composition that includes a polymer and optionally a carrier. The composition can be used to prevent or mitigate risk of infection, such as SSIs. Exemplary compositions exhibit better adhesion to skin or other surfaces compared to surgical drapes, thus reducing or eliminating potential infectionse.g., infections cause by removal of a surgical drape. Further, use of the compositions can reduce waste (e.g., by replacing surgical drapes) and costs associated with surgical procedures and with SSIs that might otherwise occur. In addition, use of exemplary compositions, as described herein, can reduce a surgical prep time and surgical inventory requirements. Further, exemplary compositions are non-leachingin contrast to some antibacterial materials that are used with typical surgical drapes.

(17) In accordance with further exemplary embodiments of the disclosure, the composition is a water-soluble material (or soluble in another diluent, such as ethanol) below the composition's gel transition temperature and maintains a solution state at low temperature whilst turning into a physical gel upon heating (e.g. upon contact with human skin). This property allows the composition to be easily sprayed on the patient's skin and form a layer of antimicrobial gel on the skin surface. During use, (e.g., during surgery) the gel might dry out and form a film on the skin; however, the film can maintain its antimicrobial activity even if dried. Moreover, since the composition returns to solution state at low temperature, the composition can be easily removed by washing with cold water (or cold alcohol) without the risk of epidermal cell layer detachment.

(18) In accordance with various aspects of exemplary embodiments, the composition can transition from a gel to a liquid state at a low temperature environment (e.g., 15 C., 20 C., 25 C., 28 C., or 30 C. or less). Exemplary compositions are designed to remain in place after application until removed with a cold solution. For example, the composition can remain in place for a period of about 1 to about 12 hours.

(19) Exemplary antimicrobial reverse thermal gel compositions include a polymer having an antimicrobial functional group, wherein the antimicrobial reverse thermal gel composition is liquid at a temperature below a skin temperature in an operating room or, e.g., 15 C., 20 C., 25 C., 28 C., or 30 C. or less and the antimicrobial reverse thermal gel material transitions to a gel at a temperature just below the skin temperature or about 25 C., 28 C., or 30 C. or more. In accordance with additional or alternative embodiments, an antimicrobial reverse thermal gel composition includes a copolymer comprising an antimicrobial functional group block and a reverse thermal gel block. Alternatively, the polymer can include, for example, a branched polymer.

(20) An exemplary polymer includes a polyuria-polyurethane polymer backbone that is functionalized with a reverse thermal gel group and an antimicrobial group. The reverse thermal gel block can include, for example, a PNIPAAm functional group to facilitate formation of a reverse thermal gel. The antimicrobial functional group block can include, for example, one or more quaternized amine groups. In accordance with other exemplary embodiments, the polymer includes a branched polymer having a reverse thermal gel group (e.g., having a PNIPAAm functional group) and an antimicrobial group (e.g., one or more quaternized amine groups).

(21) The polymers including the quaternized amine groups can kill bacteria and other microbes on contact. The bactericidal action mechanism of the quaternized amine compounds (polymers) is thought to occur through interactions between positively charged polymer sites and the negatively charged bacteria cell wall. The cationic sites of the quaternary ammonium compounds are adsorbed onto anionic sites of the cell-wall by electrostatic interaction, followed by a significant lipophilic component promoting diffusion through the cell wall. Acting as a surfactant, the long hydrophobic alkyl chains are able to disrupt the cytoplasmic membrane and cause the release of electrolytes and nucleic materials leading to cell death. As set forth in more detail below, exemplary compositions are effective at killing various microbials, including Staphylococcus aureus subsp. aureus Mu3 (MRSA/hetero-VISA), Staphylococcus epidermidis, E. coli., and the like.

(22) An exemplary polymer formulation is illustrated in FIG. 2, where, for example, x ranges from about 17-350, or about 17 to about 330, or about 17 to about 310; y ranges from about 18-61, or about 18 to about 51, or about 20 to about 41; n ranges from about 15 to about 900, or about 15 to about 800, or about 15 to about 400 and/or m ranges from about 6-22, or about 6 to about 20, or about 6-18. A percent of PNIPAAm conjugation can range, for example, from about 5 percent to about 55 percent, or about 10 to about 50 percent, or about 10 to about 45 percent.

(23) Exemplary molecular weights of the polymer blocks and the overall copolymer are presented below.

(24) Exemplary range of repeating unites of PNIPAAm (A) 17-310

(25) Molecular weight of NIPAAM (B): 113.16 g/mol.

(26) Exemplary range of molecular weight of PNIPAAM block: 2,000-35,000 g/mol.

(27) Exemplary range of repeating units of PSHU (C): 20-41.

(28) Molecular weight of PSHU (D): PSHU de-protected (no BOC groups): 489.57 g/mol.

(29) Exemplary range of molecular weight of PSHU block prior to quaternization: 10,000-20,000 g/mol (by GPC: Mn=9,725 g/mol, Mw=12,512 g/mol).

(30) After quaternization with three alkyl chains attached to PSHU.

(31) Alkyl Chain length: 6-18.

(32) Alkyl Chain molecular weight (E): 86.18-254.5 g/mol.

(33) $\begin{array}{c}\mathrm{Molecular}\mathrm{weight}\mathrm{of}\mathrm{PNiPAAM}\\ \mathrm{component}.fwdarw.i\end{array}\begin{array}{c}\mathrm{Molecular}\mathrm{weight}\mathrm{of}\mathrm{PSHU}\\ \mathrm{component}.fwdarw.\mathrm{ii}\end{array}\begin{array}{c}\mathrm{Molecular}\mathrm{weight}\mathrm{of}\mathrm{Alkyl}\mathrm{chains}\\ \mathrm{component}.fwdarw.\mathrm{iii}\end{array}$$\mathrm{Total}\mathrm{MW}\mathrm{of}\mathrm{QuatPSHUPNIPAAM}={\left[nCAB\right]}_i+{\left[CD\right]}_{\mathrm{ii}}+{\left[3E\left(\left(1-n\right)C\right)\right]}_{\mathrm{iii}}$

(34) FIG. 6 illustrates another exemplary polymer composition. In accordance with exemplary aspects of the exemplary polymer illustrated in FIG. 6, n ranges from about 2 to about 60, and/or y ranges from about 15 to about 900. Although illustrated with particular quaternized amine groups, other amine groups can additionally or alternatively be quaternized. A conjugation of the branched polymer can range from about 30% to about 60% or about 20% to about 30%.

(35) As the antimicrobial reverse thermal gel composition transitions from a liquid to a gel, the viscosity of the composition changes. For example, the viscosity of the composition before transition to a gel can ranges from less than 50 cp, less than 20 cp, or less than 10 cp at a temperature of 15 C., 20 C., 25 C., 28 C., or 30 C. or less or below a skin temperature prior to or during surgery. Additionally or alternatively, the composition can have a relatively high viscosity of greater than 50 cp, greater than 100 cp, or greater than 200 cp at a temperature of about 25 C., 28 C., or 30 C. or more, or at or slightly below a skin temperature prior to or during surgery.

(36) An amount of time for the composition to gel and to return to liquid can vary in accordance with various factors, including a percent of polymer in the composition. Table 1 below illustrates gelation times for PSHU-PNIPAAm with about 15% PNIPAAm conjugation at a temperature of about 20 C. at various polymer concentrations in a carrier comprising water.

(37) TABLE-US-00001 Weight % of polymer in solution Time to Gel (s) Time to De-Gel (s) 10% 9.4 21.9 9% 6.2 11.3 8% 4.5 15.7 7% 8.1 15.7 6% 12.6 11.6 5% 18.4 Insrant

(38) FIG. 3(A) illustrates thermal gelling properties of an exemplary aqueous composition at a low temperature, and (B) illustrates the composition after a transition to a gel at 37 C. In the illustrated case, the composition maintained its gel status over a broad range of temperature from 32 C.-45 C.

(39) Compositions in accordance with exemplary embodiments can include the polymer dispersed in a carrier. In these cases, the carrier can include a diluent, such as water or alcohol. By way of examples, the compositions can comprise, consist of, or consist essentially of about 2 to about 20, about 5 to about 15, or about 8 to about 10 weight percent polymer in a carrier, and the carrier can comprise, consist of, or consists essentially the diluent. Exemplary diluents include water and ethanol.

(40) Exemplary polymers can be formed using the following steps: 1) synthesis of PSHU using urea, N-BOC-serinol and a diisocyanate (e.g., hexamethylene diisocyanate), 2) conjugation of PNIPAAm-COOH onto PSHU, which results in PSHU-NIPAAm (in this step, the BOC protecting group in PSHU is de-protected to primary amines (NH.sub.2), and 3) quaternization of NH.sub.2 (e.g., using hexyl bromide (Br(CH.sub.2).sub.6H)). Exemplary compositions can be formed by forming the polymer as described herein and mixing the polymer with a suitable carrier.

(41) Exemplary diisocyanates suitable for use in synthesizing quaternized polyurethanes in accordance with the present invention include, but are not limited to isophorone diisocyanate, 1,4-phenylene diisocyanate, 1,3-phenylene diisocyanate, toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, 2,4,6-trimethyl-1,3-phenylene diisocyanate, tetramethylene diisocyanate, octamethylene diisocyanate, 1,3-bis(1-isocyanato-1-methylethyl) benzene, 4,4-methylenebis(phenyl isocyanate), 3,3-dimethyloxy-4,4-bisphenylene diisocyanate, 1,8 diisocyanateoctane, ,,,-tetramethyl-1,3-xylylene diisocyanate, and 3,3-dimethoxy-4,4-biphenylene diisocyanate.

(42) In accordance with additional exemplary embodiments of the disclosure, exemplary polymers are formed by 1) conjugation of PNIPAAm-COOH onto PEI, which results in PEI-NIPAAm 2) quaternization of NH2 (e.g., using hexyl bromide (Br(CH2)6H)). Exemplary compositions can be formed by forming the polymer as described herein and mixing the polymer with a suitable carrier.

(43) Antibacterial Tests

(44) FIG. 4 illustrates antibacterial activity of compositions formed in accordance with exemplary embodiments of the present disclosuree.g., the polymer composition illustrated in FIG. 2. In the illustrated example, bacteria (10.sup.4 S. aureus) were cultured under 2D and 3D conditions for 24 hr and the bacteria numbers were calculated using optical density data. For 2D culture, 10% (wt/v) polymer solution in 200 l LB was added to a 24-well plate; this solution was then placed on a 37 C. hot plate to form a gel and 500 l of the cell suspension was added. After 24 hr, the bacteria number was counted using optical density data. For 3D culture, cells were mixed with 10% (wt/v) polymer solution in 200 l LB; this solution was then placed on a 37 C. hot plate to form a gel and 500 l of LB was added. After 24 hr, the bacteria number was counted. No increase in cell number was found in both groups, while significant increase was observed in just LB. * (p>0.1) and # (p<0.001) represent statistical difference with 0 hr sample. To confirm that the polymer killed the cells after 24 hr-culture, 10.sup.4 cells were recovered from each group (named treated bacteria) and mixed with 500 l of LB in 24-well plate. After 24 hr, the bacteria number was counted. No increase was observed with cells taken from polymer culture groups, indicating that all cells were killed by the polymer. However, the culture with the cells recovered from the LB increased as much as the first culture, indicating that no cells were killed.

(45) FIG. 7-11 illustrate bactericidal properties of a composition including a polymer as illustrated in FIG. 6. In the example illustrated in FIG. 7, the polymer included about 30% PNIPAAm conjugation and the polymer was dissolved in about 10% w/v in dh.sub.2O. The antimicrobial tests conducted using Q-PEI-PNIPAAm against stationary-phase cultures of S. aureus (Staphylococcus aureus subsp. aureus Mu3 (MRSA/hetero-VISA)) showed excellent results, over an 8-log.sub.10 CFU/ml bacterial reduction. This test was conducted two more times to ensure the killing capacity of the polymer. The tests were carried out using different polymer samples and different bacteria cultures. Standard deviations were calculated, accounting for every sample. At the same time, it is also worth mentioning that PEI-PNIPAAm (30% conjugation, non quaternized) itself possesses antibacterial activity. This is due to the presence of protonated ammonium groups and non-protonated amine groups. The ethylene backbone serves as hydrophobic groups, which create repeating cationic, amphiphilic structures along the polymer backbone at a neutral pH without any further chemical modification. At the same time, it can be observed that the exemplary Q-PEI-PNIPAAm polymer exhibited a 2-, 3-, and 4-log reduction after 30, 60, and 120 minutes, which is significantly better that the PEI-PNIPAAm composition.

(46) FIG. 8 illustrates bactericidal properties of the composition of FIG. 6 with 20% and 30% PNIPAAm conjugation and the polymer dissolved 10% w/v in dh.sub.2O, evidencing that Q-PEI-PNIPAAm (20% and 30% conjugation) also exhibited an 8-log.sub.10 bacterial reduction against Staphylococcus epidermidis. Q-PEI-PNIPAAm (20% and 30% conjugation) and PEI-PNIPAAm were added to stationary-phase cultures of Staphylococcus epidermidis. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log.sub.10 CFU/ml.

(47) FIG. 9 illustrates bactericidal properties of the composition of FIG. 6 with of 30% PNIPAAm conjugation dissolved 10% w/v in dh20 against S. aureus subsp. aureus Mu3 (MRSA/hetero-VISA). Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conjugation) were added to stationary-phase cultures of Staphylococcus aureus subsp. aureus Mu3 (MRSA/hetero-VISA). Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log.sub.10 CFU/ml. Once again, the test was conducted three times using different polymer samples and different bacteria cultures. Standard deviations were calculated, accounting for every sample. In this case, PEI-PNIPAAm (30% conjugation) also exhibited an 8-log 10 bacterial reduction, but the Q-PEI-PNIPAAm significantly outperformed the PEI-PNIPAAm. Two tests showed a 6-log 10 reduction in 30 minutes, while the third test showed an 8-log reduction was achieved in the same period of time. This variability is normal, and it was concluded that in 1 hour, the polymer was able to achieve an 8-log 10 bacteria reduction in all cases.

(48) FIG. 10 illustrates results from Q-PEI-PNIPAAm and PEI-PNIPAAm (both 30% conjugation) dissolved in 10% w/v dh20 against Staphylococcus aureus. The polymer compositions were added to log-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log 10 CFU/ml. Some antibiotics require the cells to be multiplying in order to kill the bacteria. The mechanism of action of this polymer does not require this. But it may be important to ensure that the polymer can still be effective when subjected to continuously dividing cells. As can be observed in FIG. 10, Q-PEI-PNIPAAm (30% conjugation) achieved a 6-log bacteria reduction in 30 minutes.

(49) FIG. 11 illustrates antibacterial results from Q-PEI-PNIPAAm (30% and 20% conjugation) and PEI-PNIPAAm (30% conjugation), each dissolved in 10% w/v dh20 against Staphylococcus aureus.

(50) Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria. Antibacterial properties of Q-PEI-PNIPAAm (30% conjugation) were confirmed against gram-positive bacteria. Antibacterial properties of Q-PEI-PNIPAAm (30% conjugation) were then tested against gram-negative bacteria. Gram-positive bacteria generally have a thick cell wall, containing several layers of peptidoglycan, while gram-negative bacteria contains a similar cell wall, but is also surrounded by an outer membrane. This outer membrane can be harder to destabilize at times. Although gram-positive bacteria are prevalent on the skin, gram-negative bacteria can also be present in a clinical setting, and due to this, the polymer antibacterial activity against E. coli was assessed. Q-PEI-PNIPAAm (30% conjugation) exhibited a 2-, 3-, and 8-log 10 bacteria reduction after 30, 60, and 120 minutes, respectively. The lower bactericidal action, when compared to the previous tests, might be explained from the cell wall structural differences already mentioned between gram-positive and gram-negative bacteria. Q-PEI-PNIPAAm (20% conjugation) was also tested in this case, but only exhibited slightly higher bactericidal action. On the other hand, one of the controls, PEI-PNIPAAm (30% conjugation), had very little antibacterial activity, similar to the effect it had over the Mu3 strain of S. aureus.

(51) FIG. 12 illustrates Cytotoxicity Tests of Q-PEI-PNIPAAm. An MTT assay was conducted to assess the biocompatibility of Q-PEI-PNIPAAm. Several methods were used to conduct this test. Previously, the Q-PEI-PNIPAAm polymer had been placed on top of the cells mixed with the media; however, due to the gelling nature of the sample the polymer was not in contact with the cells for long period of time. Due to this, the methodology was modified and the cells were plated on top of the polymer and then trypsinized and re-plated before conducting the test; however, trypsinizing, centrifuging and re-plating cells can lead to higher variability in number of cells per well and since the cells were not left to attached on the plates for long enough the results may be inaccurate. Finally the polymer was dissolved in alcohol, left to evaporate and then the cells were plated on top. Since the MTT measures absorbance, blanks were required for each sample. However, the layer of polymer on the plate needed to be very thin so the polymer concentration tested was 1, 3 and 5%. Q-PEI-PNIPAAm (20% Conjugation) was chosen for the tests, since it had a relatively high number of quaternized amine groups and was assumed to be relatedly more toxic. Chlorhexidine 2%, a common skin preparation solution was tested as a control.

(52) FIG. 12 illustrates that no statistical difference was observed between negative control and cells exposed to PNIPAAm and PEI-PNIPAAm. A statistically significant difference was observed between the experimental samples, negative and positive control (cells cultured with 5% DMSO) (***indicates p>0.05, and **indicates p<0.05.). No statistical difference between cells exposed to chlorhexidine (FDA approved) and Q-PEI-PNIPAAm was observed. The cells exposed to Q-PEI-PNIPAAm showed statistically significant difference in metabolic activity when compared to cells cultured with complete growth medium alone and to the ones exposed to 5% DMSO. Hence, a reduction in metabolic activity was observed but the same difference was obtained in the samples with cells subjected to an FDA approved skin preparation, chlorhexidine 2%.

(53) An exemplary system in accordance with further embodiments of the disclosure includes a container (e.g., a spray bottle) and a composition as described herein. Exemplary use of the composition and/or system includes providing an antimicrobial reverse thermal gel composition as described herein and applying the composition to a surfacee.g., using a spray applicator or bottle. The composition can be applied to a surface, such as skin prior to a surgical procedure to reduce or mitigate SSIs.

(54) The present invention has been described above with reference to a number of exemplary embodiments and examples. It should be appreciated that the particular embodiments shown and described herein are illustrative of the preferred embodiments of the invention and its best mode, and are not intended to limit the scope of the invention. It will be recognized that changes and modifications may be made to the embodiments described herein without departing from the scope of the present invention. These and other changes or modifications are intended to be included within the scope of the present invention.