Injectable scaffold composition and related methods

Abstract

A composition comprising polymer particles and a carrier, wherein the polymer particles are a mixture of at least a first polymer and a second polymer, wherein the first polymer is at least partially soluble or dispersible in the carrier, and wherein the polymer particles are arranged such that they can join together to form a scaffold of polymer particles, and wherein the composition is administrable to a human or non-human animal.

Claims

1. A method of producing a scaffold in vivo in a human or non-human animal, comprising: (a) forming a scaffold by combining polymer particles capable of cross-linking with an aqueous carrier to form a composition that is administered in vivo in a human or non-human animal prior to the hardening of the scaffold, wherein the polymer particles comprise a first polymer and a second polymer, wherein the first polymer is a plasticizer having a molecular weight of 800 Da or less and is soluble or dispersible in the carrier, such that 1 wt % or more of the first polymer leach into the carrier within 20 hours at 25 C. immediately after combining the first and second polymers with the carrier, wherein the second polymer is an amorphous or semi-crystalline polymer, wherein the amount of the first polymer present in the polymer particles is from 1 to 20% by weight and the ratio of polymer to carrier is from 4:1 to 1:4, wherein the polymer particles have a glass transition temperature lower than the glass transition temperature of the second polymer on its own, wherein the polymer particles cross-link in vivo by one or more of fusion, adhesion, cohesion, and entanglement to form a scaffold of polymer particles; and wherein the scaffold is formed in vivo by removal of the first polymer from the scaffold of polymer particles by leaching, which results in a hardened scaffold structure.

2. The method of claim 1, wherein the polymer particles have a glass transition temperature of 45 C. or less.

3. The method of claim 2, wherein the polymer particles have a glass transition temperature of 37 C. or less and wherein the glass transition temperature of the polymer particles is lower than the glass transition temperature of the second polymer particle on its own.

4. The method of claim 1, wherein the plasticizer having a molecular weight of 800 Da or less is selected from the group consisting of: polyethylene glycol (PEG), poly(propylene adipate) (PPA), polyt(butylene adipate) (PBA), poly lactic acid (PLA), polyglycolic acids (PGA), poly(D,Lvlactide-co-glycolide)(PLGA), poly propylene glycol, poly capralactone, polyethylene glycol polypropylene block co-polymers.

5. The method of claim 4, wherein the plasticizer having a molecular weight of 800 Da or less is PEG.

6. The method of claim 5, wherein the plasticiser is PEG having a molecular weight of 400 Da or less.

7. The method of claim 1, wherein the amount of the first polymer present in the polymer particles is from 3% to 10% by weight.

8. The method of claim 1, wherein the second polymer is selected from the group comprising poly lactic acid (PLA), polyglycolic acids, poly(D,L-Iactide-co-glycolide)(PLGA), poly D,LIactic acid (PDLLA), poly-Iactide poly-glycolide copolymers and combinations thereof.

9. The method of claim 7, wherein the first polymer is PEG and the second polymer is PLGA.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates the rheological profiles of PLGA/PEG scaffolds after fabrication in (a) humid conditions where PEG leaches out of the polymer particles and (b) non-humid conditions where PEG does not leach out of the polymer particles;

(2) FIG. 2 illustrates the relative compressive strengths of PLGA/PEG scaffolds after fabrication in both humid and non-humid conditions;

(3) FIG. 3a is a mass spectra showing the leaching of PEG from the scaffold, which shows the quantity of PEG remaining in the scaffold at each time point, in relation to PEG 400 ions with mass of 300.2 Da;

(4) FIG. 3b is a mass spectra showing the leaching of PEG from the scaffold, which shows the quantity of PEG remaining in the scaffold at each time point, in relation to PEG 400 ions with mass of 413.2 Da;

(5) FIG. 3c is a mass spectra showing the leaching of PEG from the scaffold, which shows the quantity of PEG remaining in the scaffold at each time point, in relation to PEG 400 ions with mass of 613.2 Da;

(6) FIG. 4 illustrates the compressive strength values for PLGA scaffolds containing 10% PEG at three different molecular weights after being submerged for 24 hours in distilled water.

EXAMPLE 1PRODUCTION AND TESTING OF SCAFFOLDS

(7) a) Scaffold Production

(8) Microparticles fabricated from poly(lactic-co-glycolic acid) and the plasticizing agent poly(ethylene glycol) (5%) were combined with a saline solution (0.9%) in a 1:1 ratio and the mixture extruded from a syringe into a plastic sealable bag pre-heated to 37 C. The bag was sealed and placed in an oven for 2 hours to form scaffolds, before removal of the scaffold and analysis. The bag mimicked the in vivo environment by retaining moisture resulting in a more humid environment. The experiment was repeated but without using the bag, thus acting as a control (non-humid environment).

(9) b) Rheological Assessment of Particles

(10) Scaffolds fabricated as in part a) were broken up into particulate form and dried overnight, prior to rheological assessment. The thermal profile of both particulate materials (humid and non-humid) along with naked PLGA was assessed with a rheometer (Anton Parr PhysicaMCR 301) using a parallel plates geometry. The samples (0.5 g) were assessed by using an oscillation test (0.1% strain) to measure the change in phase angle (tan delta) as the material was heated from 4 C. to 90 C.

(11) The results of the rheological assessment are shown in FIG. 1.

(12) c) Mechanical Testing of Scaffolds

(13) Scaffolds were prepared in humid and non-humid conditions as in part a). A texture analyzer (Stable Micro Systems TX.HD plus was used to assess the relative compressive strength of scaffolds fabricated in both the humid and non-humid conditions. A load of 50 kg was applied to each scaffold at a cross head speed of 1 mm/sec using a 10 mm diameter probe at room temperature. The compressive yield strength was recorded in megapascals (MPa) for each scaffold using a plot of stress Vs strain by taking the first fracture peak as the yield point.

(14) The results of the compressive strength testing are shown in FIG. 2.

EXAMPLE 2PEG LEACHING QUANTIFICATION

(15) a) Sample Preparation

(16) PLGA scaffolds prepared in non-humid conditions (n=3) and containing 5% PEG were suspended in distilled water (1 ml per scaffold) and kept in an incubator at 37 C./5% CO.sub.2. At specific time points the water was aspirated from the scaffolds, stored at 4 C. for subsequent analysis and replaced with fresh. The time points used were as follows: 30 min, 1 hr, 2 hrs, 4 hrs, 8 hrs, 24 hrs, 48 hrs, 72 hrs, 96 hrs, 7 days, 10 days, 14 days.

(17) Each 1 ml aliquot was analyzed using mass spectrometry (MS) to assess the concentration of PEG in each and thus the quantity of PEG remaining in the scaffold at each time point. A calibration curve was prepared using known concentrations of PEG in distilled water with a further 1 in 1000 dilution required for each sample due to the sensitivity of the MS instrument.

(18) b) Mass Spectrometry

(19) Mass spectrometry (MS) on the known and unknown PEG concentrations was carried out using a Waters Quattro Ultima mass spectrometer (Agilent 1100 system with a binary pump and degasser). The samples were monitored for one minute in SIR (selected ion recording) mode to detect PEG 400 ions with the following masses: 300.2, 413.2 and 613.2 Da (with a span of 0.01 Th each at a cone voltage of 35V). Cone gas was set at 701/hr and desolvation at 5201/hr. The source temperature was 125 C. and the desolvation temperature was 350 C.

(20) A carrier solution of 20% water, 80% Acetonitrile and 0.1% formic acid was used to introduce the PEG samples into the MS. The following flow profile was used to ensure smooth peak shape and efficient removal of sample from the system: 200 l/min between 0-0.2 minutes increasing to 500 l/min at 0.2 minutes and maintained at 500 l/min for 0.8 minutes.

(21) The mass spectra are shown in FIGS. 3a to 3c.

(22) Conclusions

(23) When comparing the rheological profile of the PLGA/PEG scaffold kept in dry conditions with that kept in humid conditions there is a shift to the left between 20 and 40 C., indicating additional leaching of PEG from the PLGA material under humid conditions.

(24) This additional leaching of the PEG is considered to be a contributing factor for the increased compressive strength of the scaffold produced in humid conditions when compared to that fabricated in non-humid conditions.

(25) Data from the MS shows that the majority of the PEG released does so within the first 24-48 hours (burst release). This burst release is characteristic of porous scaffolds whereby the pores act as channels through which the PEG trapped at the surface of the scaffolds can diffuse out into the surrounding water. The rate of PEG leaching is dependent upon mass with the 302.2 spectra demonstrating the most rapid release and the 613.2 spectra demonstrating the slowest release. Crucially, both the 302.2 and 413.2 spectra confirm that PEG is leaching out of the material in the short time frames (0-2 hours) that were used to fabricate the scaffolds in humid conditions.

EXAMPLE 3EFFECT OF MOLECULAR WEIGHT OF PEG

(26) a) Scaffold Fabrication

(27) Three molecular weights of PEG were used; PEG 200, 300 and 400. Nine scaffolds were fabricated to give n=3 for all three formulations.

(28) A blend of PLGA (35 KDa) and PEG (10% loading) was created with each of the three PEG molecular weights using the melt blending method.

(29) The blended material was ground into microparticles, which were then sieved into a 200-350 m size fraction. To fabricate each scaffold, 300 mg of each PLGA/PEG blend for each formulation was combined with 0.35 cc of saline. The material was packed into a PTFE mould (12 mm6 mm) and sintered for 2 hours at 37 C.

(30) After fabrication, the scaffolds were retained in the moulds (but with the bases removed) removed from the plastic bags and submerged in 30 ml of distilled water inside a 100 ml plastic container. The containers were sealed and retained in the oven for a further 24 hours at 37 C.

(31) b) Mechanical Testing

(32) Compressive testing was carried out using a TA.HD+ texture analyzer (stable micro systems) to determine the force required to fracture each scaffold at increasing strain.

(33) Each scaffold was compressed with a 50 kg load at a crosshead speed of 0.04 mm per/sec using a 10 mm diameter delrin probe to apply the force. All testing was carried out at 37 C. using a temperature controlled chamber.

(34) The results are shown in FIG. 4. The value for each fracture point is given as the compressive strength of the scaffold in mega pascals (MPa).

(35) Conclusions

(36) It can be concluded that the compressive strength of the scaffolds increases with decreasing PEG molecular weight. This is believed to be a result of the smaller PEG molecules leaching more rapidly from the scaffolds resulting in an increase in the glass transition temperature of the PLGA.