Patent Application: US-201113272874-A

Abstract:
this invention describes novel methods for fabricating nano / micro particles and capsules through template decomposition which incorporates the to - be - encapsulated molecules which are precipitated in pores of particles or in solution , i . e . below their isoelectric point , drying or by solvent adjustment methods . the encapsulation process can be followed by a deposition or adsorption of a protective shell that regulates release of the encapsulated material . the encapsulation , inclusion , manipulation , and release of various materials and bio - materials is to be conducted by delivery vehicles which are particles and capsules with sizes in the range of nanometers and micrometers . they can possess multicompartment and anisotropic geometries and can carry one or several types of various molecules . this invention can potentially be used for controlled delivery , manipulation , and release in a variety of applications requiring delivery vehicles such as cell cultures , in - vivo , subcutaneous incorporation , injection , spray - inhalation and planar surfaces , films , and stents .

Description:
we report a new method for fabrication of pure micrometer - sized microspheres . the non - toxic nature of these uniform and relatively monodisperse caco 3 templates , high loading capacity , low price , and easy preparation and mild decomposition conditions stimulated utilization of the cores for template - assisted synthesis to produce biologically active polymeric capsules ; multicompartment and stimuli - responsive capsules ; capsules loaded with material of different nature such as organic solvents , pharmaceuticals , enzymes , dna , phospholipids and polysaccharides . other cores can also include silica , polystyrene , etc . fig1 shows a scheme of microsphere fabrication in one step , i . e . without any additives . regions of stability of caco 3 templates ( soluble at acidic ph or in the presence of complexation agents , such as edta ) and proteins are also shown . if protein solution is titrated with hcl ( hydrochloric acid ) in the presence of caco 3 templates starting from ph 9 . 5 and ending with ph 5 . 2 one can observe several intermediate states ( fig1 ). in the vicinity to the isoelectric point the solubility of proteins is dramatically decreased as they become more non - polar in the polar solvent ( water ) due to the decrease of the net charge of aminoacids . the decrease of the ph below 8 . 0 induces colloidal instability of proteins that promotes protein flocculation in the pores of caco 3 microcores ( a - b ). adsorption of protein molecules on the surface of calcium carbonate promotes surface - mediated nucleation which results in the growth of insoluble protein agglomerates in the cores but not in bulk solution . non - ionic surfactants behave similar aggregating on glass surfaces [ 12 ] . during titration at acidic ph the caco 3 core is decomposed ( b - c ) followed by shrinkage of the porous protein matrix to more compact protein microspheres / beads ( c - d ). the shrinkage is driven by water removal from the pores in the protein matrix , which were created after decomposition of the caco 3 template . at the final ph value coinciding with the protein pi ( zero net charge of aminoacids ), protein - protein interaction is established mostly by hydrophobic interactions which promotes water removal from the pores and particle contraction . since core decomposition occurs at mild conditions and at strong interprotein interaction it does not induce destruction of the protein matrix ( fig1 c ) as found by analysis of the protein content in the supernatant after core decomposition . an internal structure of caco 3 can be seen in fig2 a . in contrast , protein microspheres are compact beads , fig2 c . due to shrinkage of the pores in the protein matrix , protein is homogeneously distributed in particles , fig2 b , d . protein loading in the microspheres has limits . below the initial protein / caco 3 weight ratio 2 % the spheres are not formed , most likely because the stability of the protein matrix in the cores is not high enough to compensate the high osmotic pressure created during caco 3 core dissolution . a maximum of the loading capacity is reached at a ratio 8 - 10 % ( fig3 a , depicted by a broken line ) that induces an appearance of the protein precipitates in solution together with the microspheres ( fig3 b , c ; bulk precipitates depicted by arrows ). shrinkage of protein particles takes place after core removal when there is no barrier to prevent collapsing of the porous protein matrix . the contraction extent is considerably increased with decrease of protein loading into the caco 3 templates . this can be related to a release of larger amounts of water from the more porous and hydrated protein matrix formed at lower protein loading . the collapsed protein matrix , however , contains a significant amount of water that is independent on initial protein loading into the caco 3 cores . the protein density in particles was observed to be around 0 . 3 g / cm 3 for cores loaded with protein at protein / caco 3 ratio from 2 to 15 %. the above described methods work has been shown for several biomolecules , including insulin which has isoelectric point around 5 . 3 [ 11 ] . precipitation of proteins by ph was reported not to affect the structure of proteins . [ 14 ] fig1 shows microsphere fabrication without any additives and in one step . regions of stability of caco 3 microcores ( soluble at acidic ph ) and insulin ( insoluble in ph range 4 . 5 - 7 . 5 ; see the supporting information ) have been identified . if the insulin solution is titrated with hydrochloric acid in the presence of caco 3 microcores starting from ph 9 . 5 and ending with ph 5 . 2 , a few intermediate states are observed ( fig1 ). in the vicinity of the isoelectric point ( pi of insulin 5 . 3 [ 11 ] ), the solubility of insulin is dramatically decreased because it becomes more nonpolar in the polar solvent ( water ) woing to the decrease of the net charge of aminoacids . the decrease in ph below 8 . 0 induces a colloidal instability of insulin that promotes protein flocculation in the pores of caco 3 microcores ( fig1 a , b ). adsorption of protein molecules on the surface of calcium carbonate promotes surface - mediated nucleation , which results in the growth of insoluble protein agglomerates in the cores but not in bulk solution . non - ionic surfactants behave similarly , aggregating on glass surfaces . [ 12 ] the caco 3 core is slightly negatively charged ( ζ potential of about − 8 mv ) [ 5a ] under these conditions ( ph 9 . 0 ), which does not prevent protein adsorption ( insulin is also negatively charged ) on the microcores followed by exclusive precipitation in the pores of the microcores at ph values lower than 9 . 0 . during titration at acidic ph , the caco 3 core is decomposed ( fig1 b , c ) followed by shrinkage of the porous insulin matrix to more compact protein microspheres / beads ( fig1 c , d ). the shrinkage is driven by water removal from the pores in the protein matrix ; these pores were created after decomposition of the caco 3 template . at the final ph value , which coincides with the insulin pi ( zero net charge of aminoacids ), protein — protein interactions are established mostly by hydrophobic interactions , which promotes water removal from the pores and particle contraction . as core decomposition occurs under mild conditions and with strong interprotein interactions , it does not induce destruction of the protein matrix ( fig1 c ), as found by analysis of the protein content in the supernatant after core decomposition . a highly developed internal structure of caco 3 microcores can be seen in fig2 a . in contrast , the insulin microspheres are compact beads ( fig2 c ). due to shrinkage of the pores in the protein matrix , insulin is homogeneously distributed in the microspheres ( fig2 b , d ), at least on the scale of around 30 nm that corresponds to the pore size of caco 3 cores . [ 5b ] insulin loading in the microspheres has an upper and a lower limit . below the initial protein / caco 3 weight ratio of 2 %, the microspheres are not formed , probably because the stability of the protein matrix in the cores is not high enough to compensate the high osmotic pressure created during caco 3 core dissolution . a maximum of the loading capacity is reached at a ratio of 8 - 10 % ( fig3 a , depicted by a broken line ) that induces an appearance of the protein precipitates in solution together with the microspheres ( fig3 b , c ; bulk precipitates depicted by arrows ). taking into account the low content of fitc - labeled insulin molecules ( 10 %) mixed with unlabeled insulin and the low protein density in the microspheres with relatively homogeneous protein distribution , a distance between fluorescein molecules of longer than 10 - 15 nm can be estimated . self - quenching , which takes place at interdye distance comparable to the förster distance ( 4 . 2 nm for fluorescein [ 13 ] ), is thus excluded and the fluorescence intensity is therefore proportional to the dye ( that is , protein labeled with the dye ) concentration . shrinkage of insulin microspheres takes place after core removal , when there is no barrier to prevent collapsing of the porous protein matrix . the contraction extent is considerably increased with a decrease of protein loading into the caco 3 microcores ( fig4 a ). this effect can be related to a release of larger amounts of water from the more porous and hydrated protein matrix formed at lower protein loadings . the collapsed protein matrix , however , contains a significant amount of water that is independent on the initial protein loading into the caco 3 cores . the protein density in insulin microspheres was found to be around 0 . 3 g cm − 3 for cores loaded with protein at protein / caco 3 ratios of from 2 to 15 % ( fig4 b ). the high water content is not surprising , because insulin molecules are not crystalline and rather amorphous , as shown by small - angle x - ray scattering ( saxs ; see supporting information ). amorphous insulin could have some advantages compared to a crystalline phase . bailey et al . reported that isoelectrical precipitation does not affect the secondary structure of insulin ; [ 14 ] in general , changes in secondary structure are expected to be less pronounced for the more hydrated amorphous form than for a compact crystalline form . the stability of amorphous insulin towards chemical degradation has been reported to be higher than that of crystalline form . [ 15 ] the calculated protein density corroborates well with findings of bailey et al ., who has demonstrated that insulin precipitated in solution at a ph value of about 5 has a density of slightly below 0 . 3 gcm − 3 and the content of crystalline insulin is around 5 %. [ 14 ] a low protein density is advantageous for pulmonary delivery in deep lungs . [ 16 ] particles prepared in this study have a geometric diameter ( d g ) from 2 to 4 μm ( fig4 a ) that corresponds to an aerodynamic diameter ( d a ) from 1 . 1 to 2 . 2 μm ( respirable range [ 17 ] ), because for spherical particles in water , d a is equal to d g multiplied by the square root of the particle density . [ 14 ] the finding that the microspheres studied herein have the same protein density as precipitates formed in bulk [ 14 ] ( fig3 b , c ) indicates that the shrinking insulin matrix ( fig1 c , d ) is relatively dynamic but not a frozen structure at the insulin pi . in conclusion , we show that pure insulin microspheres can be fabricated by protein templating at isoelectric points on decomposable porous microcores from caco 3 . the main features of the microspheres include uniform size , spherical shape , monodispersity , and no additives or harsh preparation conditions with minimal processing steps . we should stress that the effective method of preparing organic nanoparticles of defined size is not confined to insulin but is of more general applicability . inspecting fig1 , it can be seen that the crucial requirement is an overlap of the template stability and drug solubility along with solubility for a certain parameter ( here ph ) and otherwise insolubility upon template destruction . caco 3 is a suitable decomposable template for many reasons , but also many other proteins or even small drugs fulfill the conditions cited above . the features of the protein microspheres make the microspheres valuable for protein delivery and show potential to achieve high systemic bioavailability and avoid potential complications owing to the presence of additives . the approach developed herein can be generalized for many other proteins that can be precipitated at conditions under which caco 3 microcores are decomposed ( that is , acidic ph or the presence of edta ). experimental section fitc - labeled and unlabeled insulin from bovine pancreas with 0 . 5 % zinc content of was purchased from sigma ( germany ). caco 3 microtemplates were prepared according to the procedure described previously , [ 5b ] average particle diameter ( 5 . 5 ± 0 . 6 ) μm . caco 3 particles ( 10 mg ) were dispersed in insulin solution ( 15 ml ) with the ph value adjusted to 9 . 5 . the insulin content was chosen to obtain a protein / caco 3 mass ratio from 2 to 20 %. stock insulin contains 10 % ( w / w ) of insulin - fitc . the suspension was slowly titrated with 0 . 1 m hcl until ph 5 . 2 , followed by dialysis for one day ( float - a - lyser g2 dialysis tubes , cut - off 0 . 5 - 1 kda , spectra / por , usa ) against water ( 2 l ) with the ph value adjusted to 5 . 2 . the microspheres were stored at 4 ° c . as a suspension or lyophilized . all experiments were carried out at room temperature . the relative content of insulin in the microspheres was calculated using the integral fluorescence from insulin microspheres as a function of initial protein / caco 3 weight ratio . the protein density was calculated taking into account an average size , mass , and porosity of caco 3 particles [ 5b ] and also the adsorption isotherm ( fig3 a ). 30 - 40 particles were treated to determine the average cumulative fluorescence ( fig3 a ) and the microsphere diameter and protein density ( fig4 ). for details of clsm , tem , saxs , and insulin titration experiments , see the supporting information . an extension of this method of encapsulation can be used to simultaneously embed several molecules . embedding is conducted by admixing the to - be - encapsulated materials , molecules , with the template - forming materials while stirring . this is applicable to molecules with different or similar pi . in the case that the molecules have similar pi , encapsulation of two molecules at the same time occurs . in the case where the molecules have different pi , they will precipitate at different times . the molecules with higher pi will precipitate before those with a lower pi . this results in molecules with higher pi forming the outer layers of the end product . this method can further be extended to create multicompartment particles / capsules . this can be achieved by two or more outer compartments being synthesized by the same process as the core formation ( for example direct precipitation upon formation or direct adsorption from a solution or buffer onto or over the template with embedded molecules ) as described above . in this case , various molecules can be placed in the different compartments . in the event the template of the end product needs to be removed , the particles / capsules can be formed whose layers are comprised of the encapsulated materials in a desired sequence . a third extension of this method utilizes anisotropic particles / capsules . anisotropic particles / capsules can be obtained either from anisotropic templates , which can be synthesized by drastically enhancing the precipitation conditions , or from packing the preformed templates with or without encapsulated materials into a substrate . the substrate can be made as a porous support or a soft , for example gel - like , film . the obtained constructs can be removed from the support or films through either physical when removing supports ( for example deformation of the support , application of temperature , etc ) or chemical means when removing films ( for example adding a solution with acidic ph , weakening the attachment between capsules / particles and the template / film , etc ). this process can be further utilized in conjunction with anisotropic , multicompartment particles / capsules . this method can also be applied to amphiphilic molecules and block co - polymers . the inner core is formed from the molecules with the highest pi . this process results in the formation of micelle - like structured delivery vehicles . this method can also encapsulate cytokines . these are included within the interior of the particles / capsules and become available for cell signaling upon subsequent release . methods for release are described below . a final unique feature of this method applies to forming particles / capsules on planar surfaces , films , and stents . deposition of porous carbonate is achieved in the first step . following this , all steps described above can be performed . when necessary , the above drug delivery vehicles can be coated by polymers , gel - like polymers , antibodies , sol - gel coatings , oil based coatings , hydrophilic polymers , hydrophobic polymers , block co - polymers , block co - polymers with peg blocks , amphiphilic molecules , nano - composite materials , organic nanoparticles , inorganic nanoparticles , metal nanoparticles , magnetic nanoparticles , peg - containing polymers , lipids , or a combination of these or other materials . this step can be used to further control the permeability , control the release profiles , enhance imaging , inducing specific targeting / binding , or elude specific binding . release profiles are also dependent on the size of the delivery vehicles . polymeric nanocomposite coatings can be made from individual polymers and their combinations , such as poly - l - lysine , polyarginine , poly - glutamic acid , gelatin , polysaccharides , chitosan , dextran , and their derivatives . smart biodegradable polymers and nanocomposites can also form the coating . the thickness of the coating and coatings as well as the assembly conditions regulate regulates the release , which can be tuned for specific time intervals . immediate release can also be achieved through the application of external fields . external fields and stimuli can act as the catalyst releasing the capsule contents in applications requiring a specific release sequence . the hybrid organic - 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