Methods and Compositions for Maintaining the Conformation and Structural Integrity of Biomolecules

Abstract

A liquid ink composition includes a liquid phase and particles suspended in the liquid phase, the particles containing a target pharmaceutical or biological agent. The biological activity of the target pharmaceutical or biological agent is preserved upon suspension of the particles in the liquid phase. The liquid phase is capable of solidifying via a solidification process.

Claims

1. A liquid ink composition comprising: a liquid phase and particles suspended in the liquid phase, the particles containing a pharmaceutical or biological agent, wherein the biological activity of the pharmaceutical or biological agent is preserved upon suspension of the particles in the liquid phase, wherein the liquid phase is capable of solidifying to form three dimensional structures.

2. The liquid ink composition of claim 1, the particles being formed by a process comprising: preparing a solution, formed from water as a solvent, comprising: a pharmaceutical or a biological agent; and a substrate that is soluble in the solution, comprising one or more chemical species; combining the solution with an oil phase to form a water-in-oil emulsion in which the solution is dispersed in the oil phase; lyophilizing the emulsion, wherein the particles are formed prior to or simultaneously with the lyophilizing, wherein the pharmaceutical or biological agent is entrapped by the formed particles, wherein the substrate composition and the oil phase are selected so that the particles formed are suspendable in the liquid ink composition and the biological activity of the pharmaceutical or biological agent is preserved upon suspension of the particles in the liquid ink composition, and wherein one or more substances selected from the group consisting of a surfactant, a stabilizer, an emulsifier, and combinations thereof are incorporated as part of the substrate.

3. The liquid ink composition of claim 1, wherein the biological activity of the pharmaceutical or biological agent is preserved upon solidification of the material to form three dimensional structures.

4. A method of forming a liquid ink composition comprising: preparing a solidifiable composition comprising: a solution containing the target pharmaceutical or biological agent; a substrate that is soluble in the solution, the substrate having components capable of being solidified via a solidification process, the solidification process of the substrate being selected from the group consisting of vitrification, crystallization, physical cross-linking, chemical cross-linking, and combinations thereof; solidifying the substrate by means of the solidification process, thereby forming particles after or as a result of the solidification process, wherein the target pharmaceutical or biological agent within the resulting particles retains proper conformation to ultimately produce a desired effect; and incorporating the resulting particles into the liquid ink composition, the liquid ink composition including a liquid phase capable of solidifying to form three dimensional structures.

5. A method of forming a liquid ink comprising: preparing particles containing a pharmaceutical or biological agent according to the following steps: preparing a solution, formed from water as a solvent, comprising: a pharmaceutical or a biological agent; and a substrate that is soluble in the solution, comprising one or more chemical species; combining the solution with an oil phase to form a water-in-oil emulsion in which the solution is dispersed in the oil phase; lyophilizing the emulsion; wherein the particles are formed prior to or simultaneously with the lyophilizing, wherein the pharmaceutical or biological agent is entrapped by the formed particles, wherein the substrate composition and the oil phase are selected so that the particles formed are suspendable in the liquid phase of the ink and the biological activity of the pharmaceutical or biological agent is preserved upon suspension of the particles in the liquid phase of the ink, and wherein one or more substances selected from the group consisting of a surfactant, a stabilizer, an emulsifier, and combinations thereof are incorporated as part of the substrate prior to solidification in order to form the emulsion; suspending the particles in a liquid phase, thereby forming the liquid ink composition capable of solidifying to form three dimensional structures, wherein the biological activity of the pharmaceutical or biological agent is preserved upon suspension of the particles in the liquid phase.

Description

WORKING EXAMPLES

Example 1

[0028] Dissolve 5% w/v gelatin in 10 mL Type I water in a scintillation vial by magnetically stirring at 37° C. for 1 hour. Weigh out a 3:1 mass/mass mixture of Span 80 and Tween 80 into a scintillation vial. When the gelatin has completely dissolved, add 1 mL of this warm solution to a vial containing lyophilized growth factor and allow to dissolve. Maintain at 37° C. Add 15 mL of dry cyclohexane to the scintillation vial. Vortex the scintillation vial to incorporate the surfactants into the cyclohexane. Place a vial into the chilled water bath and, while sonicating, slowly add the gelatin solution/growth factor drop-by-drop using a pipette. When complete, immediately place the vessel into an environmental chamber at 8° C. and set the mixer plate to rotate at a speed of 35/70 for a minimum of 60 minutes to gel the gelatin. A water-soluble, non-toxic cross-linking system, such as N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (CDI) can be incorporated into the process to further condense and solidify the gelatin nanoparticles. Snap freeze and lyophilize the solution at the completion of gelation.

Example 2

[0029] Substitute sodium alginate for gelatin in Example 1 and add measured quantity of CaCl.sub.2 to the formed emulsion to yield a 40 mM solution with respect to the aqueous portion in order to gel the alginate. The NHS and CDI are also omitted. Freeze and lyophilize the emulsion following gelation of the alginate.

Example 3

[0030] In a scintillation vial, create a dextran solution in type I water at 6% w/vol. In another scintillation vial, weigh an appropriate mass of Tween 80 to yield a solution at 0.058 g/ml in the dextran solution. Mix appropriately. Weigh out lysozyme to be dissolved in 3.0 mL of the dextran/T80 solution to yield the final desired concentration. Transfer the appropriate volume into the scintillation vial containing lysozyme to create a 0.003 g/mL solution. Weight Span 80 into a 45 mL scintillation vial. Add an appropriate volume of filtered cyclohexane to the vial to obtain a final solution concentration of 0.0118 g/mL of the desired volume. Vortex to incorporate the surfactant. Place the cyclohexane/Span vial into a chilled water bath and immerse a sonicator probe. While sonicating, add the desired volume of the dextran 70/Tween 80/lysozyme solution to the cyclohexane/Span 80, drop-by-drop, to emulsify. Place the vial into a freezer at −20° C. and allow to slow freeze for a minimum of four hours, preferably overnight, to freeze the free-water in the dextran/lysozyme dispersed aqueous phase. Lyophilize the frozen emulsion upon completion.

Example 4

[0031] To a measured quantity of lysozyme, add 0.020 g of poloxamer P188 surfactant. Solubilize in Type I water at approximately 75 mg/mL. Add 0.96 g of PLGA to a tall scintillation vial and dissolve with 16 mL of filtered acetone. Maintain vessel temperature at 25° C. While stirring, add 500 μL aliquots of acetone slowly into the lysozyme solution until 2.5 mL have been added. Cap the vessel and hold for 60 seconds. Repeat the additions until approximately 5 mL have been added to the vessel. Cap and hold another 60 seconds. Switch to 100 μL aliquots of acetone and add while stirring. Cap the vessel and hold between additions for 30 seconds. Continue to add acetone until the solution turns and remains opalescent, indicating the protein has nanoprecipitated. Cap the vessel and stir for five minutes. Add a stir bar to the polymer solution and add the nanoprecipitate solution into the polymer solution in small aliquots. When complete, stir for an additional 5 minutes. The resultant solution should be opalescent and stable. Place a beaker containing 500 mL of pentane into a sonic water bath. Syringe deliver the polymer solution into the pentane bath, while sonicating, via a small diameter blunt needle at no more than 0.5 ml/min. When delivery is complete, allow the material to sit statically in pentane for 30 minutes to solidify the polymer. Pour off/pipette off the pentane and transfer the material into a small lyophilization vessel. Place the vessel into a vacuum oven at 37° C. for three hours (maximum vacuum) to extract the residual pentane. Prepare a dry ice/pentane bath, snap freeze the material for 15 minutes, and then lyophilize to remove residual water and solvent. Store appropriately.

Example 5

[0032] Prepare a DCM/Span 80 solution at 0.0045 g/mL, and a 3% Tween 80 solution in type I water. Solubilize lidocaine HCL in the Tween solution at the desired concentration. Prepare a 7.5% wt/v PLLA solution in dichloromethane/Span 80 using high molecular weight polymer. When complete, pipette 500 μL of the prepared lidocaine HCL/Tween 80 solution into the polymer solution to create a primary emulsion. Emulsify according to standard practice, preferably using pulsed sonication. Load the emulsion into a 10 mL rubber-free syringe, attach a small diameter blunt needle, and deliver the contents at a rate of 0.150 mL/min, 5 inches above a vessel containing approximately 600 mL of pentane. The vessel also contains a filter screen or basket to capture formed particles. When solution delivery is complete, wait 30 minutes to allow for particle solidification, and then transfer the particles into a lyophilization flask. Place the flask into a vacuum oven at 37° C. and allow the particles to dry for three hours. Load the particles into a scintillation vial or similar vessel, and immerse the vial into a pentane/dry ice bath to freeze the material. Lyophilize for a minimum of 24 hours to remove residual water and pentane.

Example 6

[0033] Prepare 30.0 ml of 0.4M AOT/isooctane volumetrically using a 50 ml centrifuge tube. Create a solution of PEGylated alginate at 74 mg/ml in pure water. Prepare CaCl.sub.2 solution at a concentration of 110 mM in filtered nanopure water. Dissolve BSA at a concentration of 7.33 mg/ml in filtered, nanopure water. Load 0.77 ml of PEGylated alginate solution into a 3 ml disposable syringe (A). Load 0.33 ml of BSA solution into another 3 ml disposable syringe (B), and load 0.11 ml of CaCl.sub.2 solution into syringe a third syringe (C). Connect syringes A&B and mix the two solutions by moving the material from one syringe to the other 20 times. Separate the two syringes and connect a new (D) 3 ml disposable syringe to the syringe containing the mixed material. Push the PEGylated alginate/BSA solution into the new syringe through a 0.2 μm filter. Dispose of the empty syringe and filter. Empty the syringe containing of filtered PEGylated alginate into a 1.5 mL centrifuge tube. Withdraw 1.000 ml from the Eppendorf, and pipette it into the vial containing the lyophilized protein to be loaded. Mix well, but do not vortex the material. Connect a sterile needle to syringe D and carefully withdraw the fluid from the vial containing the protein. Connect syringe D to syringe C, which contains CaCl.sub.2 solution. Push the PEGylated alginate/BSA/protein into the CaCl.sub.2 solution, then syringe back and forth 20 times to ensure that the Ca.sup.2+ is well dispersed. Immediately empty the syringe contents into previously prepared 20.9 ml of 0.4M AOT/isooctane in a 50 ml centrifuge tube. Cap the tube and vortex. Place the tube vertically in the refrigerator at 4° C. immediately after vortexing. Allow to gel overnight. Lightly centrifuge the tube at 1500 rpm for 15 minutes. Discard the supernatant without disturbing the pellet and then wash the retained material three times with ethanol. Remove/evaporate the ethanol and store the resulting material for use.

Example 7

[0034] Create a dextran/lysozyme solution in nanopure water, with dextran 70 at 6% w/w, and with lysozyme added at 2 mg/mL. Create a PEG 8000 solution at 6% w/w. Create a 1:10 w/w blend of the dextran 70/lysozyme:PEG 8000 solutions and vortex to blend. These ratios should allow for the creation of a single phase aqueous system. Place the solutions in the freezer at −20° C. for at least 8 hours to slow freeze and phase separate into a water-in-water emulsion. Snap freeze and lyophilize the vial for 48 hours, minimum. When complete, add 10 mL dichloromethane to the tube, vortex, and then centrifuge for 15 minutes to collect the formed dextran particles. Discard the supernatant, refill with DCM, and centrifuge again. Repeat this washing process a total of three times. Dry the tubes for at least 8 hours in a vacuum oven at RT and maximum vacuum.

Example 8

[0035] As in the previous example, create a 1:10 w/w blend of dextran 70/PEG 8000 solution containing a protein of interest. Spray atomize the single-phase solution into liquid nitrogen to create ice particles of PEG/dextran/protein that are sub-25 μm. Collect the particles while frozen and disperse into a vessel of cyclohexane chilled to 7° C. The frozen dextran/PEG/protein particles should locally freeze the cyclohexane, which has a freezing temperature of approximately 6.5° C., and remain dispersed. Immediately hard freeze the suspension in dry ice/pentane, and then allow the frozen material to return to 4° C. to thaw the aqueous portion but maintain the cyclohexane in a frozen state. Hold at this temperature for an hour, and then transfer the material to a −20° C. freezer for at least 8 hours to invoke temperature-induced phase separation of the dextran/PEG and vitrification of the dextran. The resultant glassy dextran will encapsulate and protect the protein. Hard freeze in dry ice/pentane, and then lyophilize to recover particles. Wash with DCM repeatedly to remove PEG.

Example 9

[0036] Create a solution of dextran 70 containing a protein of interest. Using a sonic atomizer, spray atomize the solution into liquid nitrogen to create ice particles of dextran that are sub-20 μm. Collect the particles while frozen and disperse into a vessel of cyclohexane chilled to 7° C. The frozen dextran/protein particles should locally freeze the cyclohexane, which has a freezing temperature of approximately 6.5° C., and remain dispersed. Immediately hard freeze the suspension in dry ice/pentane, and then allow the frozen material to return to 4° C. to thaw the aqueous portion but maintain the cyclohexane in a frozen state. Hold at this temperature for an hour, and then transfer the material to a −20° C. freezer for at least 8 hours to invoke temperature-induced vitrification of the dextran. The resultant glassy dextran will encapsulate and protect the protein. Hard freeze in dry ice/pentane, and then lyophilize to recover particles.

Example 10

[0037] Create a solution of dextran 70 at 10% w/vol in a scintillation vial and allow to solubilize at 37° C. for 30 minutes. Weigh trehalose into a scintillation vial to obtain a desired end concentration of 10% w/vol. Add an appropriate volume of the dextran solution to this vial and allow to solubilize. Measure Tween 80 into a scintillation vial to obtain the desired final volume at a concentration of 5.8% wt/vol. Add the dextran/trehalose solution to this to obtain the desired volume of aqueous solution. Prepare a Span 80/cycloheptane solution at 1.18% w/vol. To a vial containing 1 mg/mL protein, add 500 μL of the prepared aqueous solution. Mix gently to incorporate. Pipette this solution into a vial containing 7.5 mL of the cycloheptane/Span 80 solution and emulsify to create a nano-size colloidal suspension. Transfer the emulsion to a 20 mL lyophilization vial and place onto a shelf that has been pre-cooled to −55° C. Allow to freeze for 2 hours. Lyophilize the material to recover the lyocake containing nanoparticles of protein encapsulated in trehalose/dextran.

[0038] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present invention to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the preferred embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.