the present invention concerns alumina sol - gel materials comprising a labile pharmaceutically active agent , such as peptides , proteins such as hormones , enzymes , antibodies , receptor binding proteins , antibody fragments , antibody conjugates , nucleic acids such as aptamers , irna , sirna , microrna , dna , rna , antisense nucleic acid or the like , antisense nucleic acid and the like , vegf inhibitors , macrocyclic lactones , dopamine agonists , dopamine antagonists , low - molecular weight compounds , high - molecular - weight compounds , and conjugated bioactive agents . in the following , the uniqueness of the invention is demonstrated by exemplary biological models . the composite bioactive @ alumina were prepared as particles in the size range of 1 - 10 microns , similar to the particle size of alumina adsorbed / precipitated vaccines . the alumina used may be any one of the 6 alumina composites known . in the exemplary models provided herein , the alumina was polycristalline boehmite ( gamma - alumina , alo ( oh )) which is the bio - active form of alumina used in commercial aluminum hydroxide adsorbed vaccines . the crystalline form was verified using xrd for both undoped and doped alumina . estimation of the amount of entrapped bioactive , e . g ., protein was done by tga analysis , uv spectrophotometry at 280 nm and the micro - bradford assay ( 595 nm ). the highest load of entrapped protein tested was 27 % by weight , as this is the upper limit of antigen given per immunization in conventional alumina adsorbed vaccines ( 25 ug antigen per 25 ug al 3 + ). all measurements indicated a nearly quantitative entrapment of the protein . higher antigen / alumina ratios were also possible , and this may be useful in light of the limitations on the amount of aluminum per dose ( usa : 0 . 85 mg / dose , europe : 1 . 25 mg / dose ). aluminum isopropoxide , acid phosphatase ( acp , cat . no . p - 0157 ), asparaginase ( asp , cat . no . a - 0884 ), horseradish peroxidase ( hrp , cat . no p - 8375 ), l - asparagine , nessler &# 39 ; s reagent , trichloroacetic acid , ammonium sulfate , p - nitrophenyl phosphate ( pnpp ), 2 , 2 ′- azino - bis ( 3 - ethylbenzothiazoline - 6 - sulfonic acid ) ( abts ), hydrogen peroxide 30 % were all obtained from sigma - aldrich . glycine buffers were prepared from glycine solutions ( 0 . 05m ; from sigma - aldrich ) with desired volumes of 1 . 0 m naoh or 1 . 0 m hcl . ultrasonic ( us ) energy source was used to prepare alumina sol at neutral ph . in detail , 2 . 2 g of al ( c 3 h 7 o ) 3 was added to 50 ml of deionized water at 90 ° c . and a white precipitate was formed immediately . before us treatment , the precipitate was kept at 90 ° c . under vigorous stirring for 15 min to complete the production of boehmite nanoparticles and to complete the evaporation of the isopropanol formed during hydrolysis . the final suspension was ultrasonically treated ( elma s 10h 37 khz , 30 w ) for 2 h . after 2 h a viscous sol was formed . the resulting sol was cooled to room temperature . the dried matrix had a surface area of 153 m2 / g , pore volume of 0 . 097 cm 3 / g and an average pore size of ˜ 2 . 5 nm . a non - limiting example of bioactives in accordance with the invention is proteins . as model proteins , three proteins of the albumin family were used : ovalbumin ( ova ), bovine serum albumin ( bsa ) and human serum albumin ( hsa ). various entrapment procedures were used , denoted hereinbelow as i , ii , iii and iv . method i : as a first step , boehmite sol was prepared as follows : 2 . 837 g al ( i - pro ) 3 was hydrolyzed for 45 min in 51 ml ddw at 85 ° c . in an air - open flask . then , 1 . 67 ml hcl 1m was added and temperature was increased to 95 ° c . to catalyze the peptization . the flask was kept air - open for additional 3 hrs to enable isopropanol evaporation , and then placed under reflux for additional 16 hours . then , the reflux was removed and the water was allowed to evaporate by air - exposure until the total sol volume was reduced to less than 25 ml , which was then re - adjusted to 25 ml with ddw ( 15 mg al 3 + / ml sol ) ( alumina - i ). the second step — the protein entrapment — was carried out as follows : 45 . 4 mg ova grade v was dissolved in 42 ml 25 mm l - histidine buffer at ph = 6 . 91 . 4 . 0 ml of the protein - buffer solution was added to 1 . 0 ml of alumina sol , vortexed and left for 1 h under shaking in ice . then 100 μl of naf 50 mg / ml solution was added to the 5 . 0 ml protein - sol under vortex . the solution became immediately turbid and the gel forms . the tube was centrifugated for 10 min at 5 , 000 rpm and placed at 4 ° c . for 10 days for gel aging . the supernatant was then removed and the tube placed in a desiccator under vacuum for 3 - 7 days for xerogel formation , until the gel has shrunk to about 10 % its initial volume ( ova @ alumina - i ). the above ratios yielded a 7 . 2 μg ova / 25 ng al 3 + composite , which was approximately 10 % ova dopant by weight . methods ii and iii : the second and third methods used yoldas procedures by peptizing aluminum hydroxide precipitate in acids : acetic acid ( alumina - ii ) or nitric acid ( alumina - iii ). in detail , 3 . 28 g of al ( c 3 h 7 o ) 3 was added to 50 ml of deionized water and a white precipitate was formed immediately . the precipitate was peptized with 0 . 2 ml of concentrated nitric acid or with 2 . 0 ml for acetic acid at 90 ° c . under vigorous stirring for 2 h ( also to evaporate the isopropanol formed during hydrolysis ) to produce stable transparent boehmite sols , ( 6 . 7 mg and 6 . 5 mg al3 +/ ml sol , respectively ). the ph of the final solution was 2 . 8 ( 4 . 8 in acetic media ). the second step — the albumin entrapment — was carried out un - buffered by dissolving 0 . 03 g of the protein ( bsa and hsa within alumina - ii and alumina - iii respectively ) in 20 . 0 ml boehmite sol at room temperature . the suspension was aged for 3 h and dried at 20 ° c . for a week . tga analyses indicated a ˜ 8 w / w % loading in the two samples . method iv : this method ( alumina - iv ) followed method ii but without any acid or buffer . the white precipitate was ultrasonically treated ( 37 khz , 0 . 063 kw ) for 4 h ( final ph = 7 . 3 ). after 4 h a viscous sol was formed . the second step — the albumin entrapment — was carried out at room temperature by dissolving 0 . 03 g of the protein in 20 . 0 ml of the sol ( alumina - iv ): with bsa — bsa @ alumina - iv , or with hsa — hsa @ alumina - iv . the suspension was treated as above resulting in a ˜ 8 w / w % loading . in the following , protein @ alumina was prepared ( e . g ., bsa @ alumina - iv ), where the protein was ova , bsa or hsa , and methods were ( as detailed in the experimental section below ): i — catalysis with naf , ii — peptization with acetic acid , iii - peptization with nitic acid , and iv — ultrasonic preparation at neutral ph . it should be noted that the use of acetic acid and particularly of ultrasonic treatment provide solution ph values of 4 . 8 and 7 . 3 , respectively , that are suitable for biomolecules and corresponds to an optimum range at which the albumin molecules and the particles have opposite charges , providing the needed electrostatic interaction for entrapment . for verifying the entrapment of the proteins within the alumina sol - gel , the thermal behavior was tested , as described below , together with analyzing the organic - bio - organic hybrid materials under ir spectroscopy ( ftir ), which clearly showed a superposition of the typical absorption bands of the matrix and the biological dopant . a representative example — hsa @ alumina - iv — is shown in fig1 , wherein the ir spectrum of the composite — line 2 was indeed a superposition of the spectra of hsa ( line 1 ) and alumina ( line 3 ). afm pictures in the semi - contact mode of the dried samples reveal the aggregation of these elementary particles into larger particles of several tens of nm , as shown for instance for hsa @ alumina - ii ( fig3 ; as expected , dls data provided larger hydrodynamic sizes ). surface area and porosity analyses ( by nitrogen adsorption , analyzed by the bet and bjh equations ) confirmed typical microporosity , with some induced changes due to protein entrapment . the values before protein entrapment were : surface area of 246 m 2 / gr , pore volume of 0 . 23 cm 3 / gr and pore size of ˜ 4 nm , with an increase , while the surface area values after entrapment were 370 and 356 m 2 / gr for bsa and hsa , respectively , with an increase the pore volume to 0 . 34 cm 3 / gr for both proteins , and with a little change in the average pore size . it seems that the effect of the protein was to interfere with the aggregation , resulting in a more open structure . the thermal stability of proteins was a very strict limitation of their use for various medical and industrial applications , often requiring refrigeration . the use of alumina makes this potential stabilization even more relevant and interesting . the stability studies have been carried out by differential scanning calorimetry ( dsc ). dsc has been traditionally used for the follow - up of phase transitions within materials , and is less known to determine phase transitions in proteins due to the denaturation process . indeed , proteins demonstrate a dsc peak ( either the original curve or its second derivative ) upon heating . as shown in fig4 , the denaturation temperature of ova was shifted by 30 degrees when entrapped by method i after three days of aging and reached 100 ° c ., an extremely high temperature for denaturation . similar stabilization was observed for bsa @ alumina - ii and iii ( fig4 ). an exceptionally high stabilization was obtained for hsa in hsa @ alumina - iv : an increase of 54 ° c . to the denaturation temperature of 120 ° c . (!) ( fig5 b ), highlighting that the ultrasonic sol - treatment utilized in accordance with the invention , was optimal for bioactives such as proteins . fig6 shows yet another feature of the stabilization by entrapment in alumina : the stabilization of ova progresses with time of aging of the doped alumina - gel ( method i ). when no time was allocated for xerogel transformation ( 0 days ), the peak mean was only at ˜ 47 ° c . one day of xerogel transformation , and the peak mean value shifted towards 60 ° c . two days and the mean value reached 75 ° c . three days and it peaked at 100 ° c . thus , the method and materials of the invention may be regarded protein - friendly for entrapment of bioactives such as proteins . trapped in alumina , such materials exhibited marked stabilization and thus opened a potential door for sol - gel materials to be considered as carriers of bioactive molecules . in - vivo experiments in balb / c mice have demonstrated high levels of specific antibody titer against a model antigen — ovalbumin — entrapped in alumina , following immunization by injection . no side effects have been observed for the duration of the experiment ( months ), supporting the safety of the composite vaccines . some unique properties of the immune response have been observed . these include a significant igm titer 31 days following vaccination suggesting that the antigen was being released from the composite antigen - adjuvant matrix one month after administration ; and an extremely high antigen specific igg titer was observed 67 days following vaccination ( an end - point titer of 1 : 51 , 200 , with no boost ). this resembled the titer obtained by adjuvants which are far stronger than alumina — freund &# 39 ; s complete adjuvant , or three immunizations with conventional alumina adsorbed vaccines , obtained here by only a single immunization with the composite alumina entrapped vaccine . controlled release of the antigen during a prolonged period of time may provide gradually and continually exposure of the recipient &# 39 ; s immune system . this may yield improved and longer - term immunological memory . in fact , the immune response may be optimized by tailoring the desired release rate . another considerable benefit may be the reduction of the number of required vaccinations to obtain an adequate immunological memory . the use of an alumina adjuvant as a releasing matrix , adds unique features , as explained above . dozens of vaccine adjuvants which have been disqualified due to excess potency when inoculated directly ( e . g . fca ) may be reassessed under controlled release conditions when entrapped within a sol - gel matrix . one of the aforementioned benefits of the entrapment process is the gradual , controlled release rate . spectrophotometric micro - bradford assay , known as a sensitive assay to determine protein concentrations , was used to evaluate the amount of released protein from alumina entrapped gels , compared to desorption , following 12 days of incubation at ambient temperature with various concentrations of phosphate buffer . the results ( table 1 ) clearly show that the entrapped protein was released much slower as compared to the desorption of adsorbed protein . it should be stressed that while control of release from the adsorbed state was an almost impossible task , the rate of protein release from the entrapped matrix could be controlled by changing parameters of the sol - gel process ( e . g . longer aging time ; in this experiment the gels were aged for only 1 day ). 10 weeks old balb / c mice were immunized with the alumina entrapped vaccines . in order to evaluate the vaccines safety , the mice were physically examined and weighed 3 times a week in the first month following vaccination by a veterinary or a veterinary technician . after the first month , physical examination + weighing were carried out twice a week . it should be noted that the ethical committee conditioned any further testing of the vaccines in larger population of mice by demonstrating first that they were safe in a small group of mice . throughout 50 days following immunization no side effects were observed . 3 days following immunization the vaccines were completely absorbed and the mice did not show any signs of pain when touching the injection site . as can be seen in fig8 , there was a general trend of increase in all mice weight vs . time following immunization . the bleedings on days 13 and 31 were marked , usually followed by a small decrease in weight . bleedings ( 100 ul ) were taken from the submandibular vein before immunization ( day 0 ), on day 13 , on day 31 and on day 67 following vaccination . sera were obtained from blood and analyzed for igm and igg ova specific titer in comparison to day 0 of the same mouse . all sera were kept at 4 ° c . until analysis . fig7 clearly demonstrates that anti - ova specific titer was obtained in all mice , both igm and igg , thus proving the concept that alumina entrapped vaccines work . for the entrapment of acp , a mixture of 50 μl , of glycine - hcl buffer solution ( ph 4 . 5 ) and 1500 μl , of freshly prepared alumina sol was transferred to a cuvette and then 40 μl , of acp ( 200 u / ml ) was added . ten minutes later the sol was left in vacuum desiccator at room temperature for 24 hours . the acp @ alumina ( 0 . 009 % by weight for the kinetics studies and 0 . 1 % for the cd and dsc measurements ) was rinsed inside final polystyrene cuvette with 1 . 0 ml of glycine solution ( ph 4 . 5 ) to assure removal of any adsorbed protein . the entrapment of hrp within alumina ( 0 . 05 %) was carried out similarly with glycine - hcl buffer solution at ph 5 . 0 and 40 μl , of hrp ( 20 u / ml ). the entrapment of asp within alumina ( 0 . 03 %) was carried out similarly with glycine - naoh buffer solution ( ph 8 . 5 ) and 40 μl , of asp ( 152 u / ml ). the entrapment of all enzymes was full , as indicated by lack of activity in the supernatant solutions and washings ( see below ). acp @ alumina . after rinsing with 1 . 0 ml of glycine solution ( ph 4 . 5 ), the bioactive hybrid was left for incubation at 37 ° c . for 30 min . then , the rinsing solution was replaced with 1 . 0 ml of 10 . 8 mm of the substrate ( pnpp ) in glycine — hcl buffer solution ( ph 4 . 5 ), and the enzymatic activity was measured by following the formation of p - nitrophenolate ( pnp , fig1 - 1 ) spectroscopically through the absorption at 405 nm , at temperatures ranging from 30 to 75 ° c . readings were taken every 20 sec for 30 min . the rinsing solution was also tested for enzymatic activity by the transfer of 500 μl of the solutions into cuvettes and adding 1 . 0 ml of 10 . 8 mm of substrate ( pnpp ) solution . the dose — response kinetics of the composites was measured by using different pnpp concentrations . the rates for the enzymatic reaction were calculated according to regular pseudo - first order kinetics and the michaelis - menten equations . for comparative analysis of free acp , 1 μl ( 780 u / ml ) in 1 . 0 ml glycine solution ( ph 4 . 5 ) was taken and reacted similarly to the entrapped enzyme . hrp @ alumina . the enzymatic activities of free and entrapped hrp were determined using abts as the reducing substrate ( fig9 ). the assay system was a mixture of 1 . 0 ml of 0 . 5 mm abts and 0 . 2 ml of 30 % h2o2 in 2 . 0 ml of 50 mm glycine - hcl buffer ( ph 5 . 0 ). the enzymatic activity was measured spectrophotometrically as a rise in absorbance of the formation of the oxidized abts at 425 nm . to polystyrene cuvettes containing hrp @ alumina in 1 . 0 ml glycine solution ( ph 5 . 0 ) was added , and comparative incubation was carried out at 37 ° c . and 75 ° c . for 1 h . the rinsing solution of hrp @ alumina sample was also tested for enzymatic activity by the transfer of 500 μl of the solutions into cuvettes and adding 1 . 0 ml of 0 . 5 mm abts and 0 . 2 ml of 0 . 2 mm h 2 o 2 in 2 ml of 50 mm glycine - hcl buffer ( ph 5 . 0 ). the activity of hrp was determined by following the formation of oxidized abts forming during the first 10 min of the reaction . asp @ alumina . the activity was measured using nessler &# 39 ; s reagent ( fig1 ). the reaction mixture was of a final volume of 3 . 0 ml containing 50 mm glycine - naoh buffer ( ph 8 . 5 ) and 189 mm l - asparagine . this was placed in either the final polystyrene cuvette containing the asp @ alumina or added to 0 . 4 μl ( 152 u / ml ) enzyme solution . both of cuvettes were kept under 1 . 0 ml glycine - naoh solution at ph 8 . 5 during incubation at 37 ° c . and 75 ° c . for 1 h . the rinsing solution of the asp @ alumina was also tested for enzymatic activity by the transfer of 500 μl of the solutions into cuvettes and adding 50 mm glycine - naoh buffer ( ph 8 . 5 ), 189 mm l - asparagine . the reaction was stopped by adding 100 μl of 1 . 5 m trichloroacetic acid . the solution was centrifuged followed by adding 1 ml of nessler &# 39 ; s reagent to 1 ml of the supernatant . the absorbance at 436 nm of the resulting solution gave a measure of enzyme activity . a standard curve was prepared with ammonium sulfate and the activity of asp was determined by following the ammonia formation during 30 min of the reaction . testing the free enzymes . to compensate for the slower reactivity of the entrapped enzymes , lower concentrations of the free enzymes were taken —× 4 smaller for acp , and × 10 smaller for the other two enzymes . the crystal phase and crystallinity of the samples have been studied by x - ray diffraction method ( bruker d8 advance ) using cu - kα irradiation ( λ = 1 . 54 å ), samples being scanned along 2θ in the range of 4 - 75 ° at a speed of 0 . 5 degrees per minute . the spectral analysis of enzymatic activity was carried out using hp 8453 diode array spectrophotometer . for scanning electron microscopy ( sem , ultrahigh resolution magellan 400 l electron microscope ), the final suspension of the entrapped enzyme was coated on silicon wafer and fully dried under vacuum . dsc curves were obtained with 204 f1 phoenix netzsch apparatus and a heating rate of 10 ° c . min − 1 was used from 30 ° c . to 150 ° c . under nitrogen . cd spectra were recorded on a j - 810 jasco spectropolarimeter . the measurements of solutions were taken using a 2 - mm path length cuvette and coated slides were used for the entrapped proteins at 20 ° c . thermal stability studies were performed between 20 and 90 ° c ., at a constant heating rate of 3 ° c ./ min . the enzymatic activity and thermal stability of acid - phosphatase entrapped in alumina ( acp @ alumina ) fig1 shows the activity of the entrapped enzyme as a function of the substrate concentration . the activity observed was from the entrapped enzyme only : rinsing solutions have been analyzed and their activity was found to be negligible , and in fact even lower than simple non - enzymatic hydrolysis . for instance , while acp @ alumina reaches a rate of 0 . 351 mm / min after 10 min at 30 ° c ., the activities of the washing solution and of non - enzymatic hydrolysis were 0 . 011 and 0 . 014 mm / min , respectively ; similar observations characterize the two other enzyme entrapments in this report . analysis of the data of fig1 a with an eadie - hofstee plot ( fig1 b ) provides michaelis - menten ( mm ) parameters of vmax = 2 . 3 mm / min and km = 3 . 38 mmol km of free acp in glycine buffer solution is 1 . 25 mmol , and the difference between the two km values is a reflection of the diffusional difficulties that the porous alumina matrix imposes on the substrate molecules . yet the fact that the graph is perfectly linear implies that the mm mechanism is obeyed , which in turn implies that the distribution of the enzyme molecules is homogeneous within the matrix , and that there is no denaturation of the entrapped enzyme : such denaturation leads usually to a distribution of activities ( relating to the distribution of the strength of the denaturation ), which in turn leads to non - compliance with the mm mechanism . the activity is possible at all because of the porosity of the matrix . the crystalline nanorods ( about 1 × 10 nm ) morphology was densely packed . the mesopores from few to more than 20 nm were also observed . similar morphologies were obtained for hrp @ alumina and asp @ alumina . in solution the typical thermal behavior of acp was characterized by the expected gradual decrease of activity at temperatures above 400 ° c ., coming to a full stop of activity at temperatures exceeding 70 ° c . ; and it should be noted that for adsorbed acp on ca - polygalacturonate the thermal stability was even worse . in accordance with the present technology , the stability of alumina - entrapped acp was found to be so high that its activity increased with temperature up to 60 ° c . (!). decrease in activity began beyond that temperature , but still , the activity at the highest temperature measured , 750 ° c ., was higher than at room temperature by about two orders of magnitude . furthermore , another manifestation of the thermal stability of the entrapped acp was its stability to prolonged cycle periods of heating to 60 ° c . and cooling . as the enzymatic hydrolysis of pnpp was pseudo - first - order ( water is in excess ), the temperature dependence of the rate constant is describable by arrhenius &# 39 ; law , k = a · e − e a / rt , ( where k is the rate constant , ea the activation energy , t the temperature , r the gas constant and a is the arrhenius pre - factor ). the data in fig1 a was thus subjected to an arrhenius analysis , and the results are shown in fig1 a . two quite different slopes are seen : the negative slope corresponds to ( positive ) activation energy of the pnpp hydrolysis reaction of 110 kj / mol ; that is , up to 60 ° c . the classical increase of activity with temperature is observed . then there is the positive slope with an apparent negative activation energy of − 28 kj / mol , which represents the temperature range where denaturation occurs , that is , where the activity of the enzyme is disrupted by the heating . in solution ( fig1 b ) the enzymatic reaction activation energy ( measured over a temperature range of 20 - 50 ° c .) was lower — 60 kj / mol — and attributed to the higher value for the entrapped to diffusional restrictions of the pnpp inside the micropores network , until the enzyme was reached . note also that the slope representing the denaturation process was much steeper in solution with apparent negative activation energy of − 144 kj / mol . this then is the trade - off : a very high thermal stability , but lower reaction rates . for many applications , thermal stability of the enzyme is the more important feature . finally , the arrhenius pre - factors , a , of the free and entrapped enzymes were measured . a = zρ , where z is the frequency of collisions of the substrate molecule with its reactivity target , and ρ is the orientation parameter ( usually less than unity ). for the entrapped enzyme the prefactor value is a = 3 . 54 . 1014 sec − 1 , and this value is six orders of magnitude higher (!) than that of the free enzyme : 4 . 34 . 108 sec − 1 . to understand this large difference , it should be noted that the perfect linear compliance to the arrehnius equation indicates that the rate determining step is the enzymatic reaction . therefore a plausible explanation for the very high a value in the entrapped case is a dramatic increase in the number of collisions within nanopore that leads to the active site and the substrate that resides near it . furthermore , this high value probably also reflects a favorable orientation of the pnpp molecules for fruitful collisions with the active site , that is , that the nanopore acts as an orientation - directing restriction . in other words , in the nanopores , although though initial encounters are lower than in the solution , once the reactants meet the enzyme , they get trapped to provide a large number of collisions before escaping the cage . these observations can be interpreted by using protein dynamics considerations within nanopores . it is known that silica sol - gel entrapped proteins are essentially immobile within the matrix , consistent with interactions between the matrix surface functional groups and the protein surface functional groups , causing motion restriction . it suggests that the amorphous ceramic matrix is likely templated around the protein , leading to the restricted mobility . in this context , it was shown that entrapment results in only little perturbation of the tertiary and quaternary conformation of the protein . the exceptional thermal stability and dramatic increase in the number of collisions within nanopore in the present case of entrapment within alumina , leads one to suggest that the rotational mobility of enzyme within alumina is restricted even to a higher degree than in silica , providing the needed conditions for thermodynamic stability . this observation can be related to the crystalline nature of the alumina matrix . amorphous silica is relatively soft compared to alumina , and during the heating the structure can still rearrange , providing more freedom leading eventually to earlier unfolding . the dense structure of crystalline boehmite nanorods of alumina keeps enzymes tighter , preventing easy unfolding . two additional measurements were employed to investigate the thermal stability . the first of these is comparative circular dichroism ( cd ) follow - up . since the cd spectrum is sensitive to conformational changes and is a common spectroscopic method for studying enzyme structure , one can use this tool for detection of denaturation as well . as seen in fig1 a , the cd spectra of the free and entrapped enzyme are quite similar , indicating the ( non - trivial result ) that the conformation of enzyme remains intact upon entrapment . the free and entrapped enzymes were then heated from 10 to 90 ° c . at a rate of 3 ° c ./ min , and the ellipticity at the characteristic helical value of 215 nm was monitored ( fig1 b ). it is clearly evident that while the free enzyme undergoes conformational changes above 40 ° c . ( the ellipticity drops ) in the case of the entrapped enzyme , the ellipticity remains constant in the interval 20 - 90 ° c . yet another proof of the enhanced thermal stability comes from differential scanning calorimetry ( dsc ) analysis . the result of this analysis , which was developed for the above detailed entrapped protein stability indicator , follows the changes in the denaturation temperature upon entrapment . extremely high stabilization is seen in fig1 : the denaturation temperature is shifted by 51 ° c . higher . this observation is in agreement with the cd spectra and kinetic measurements analyses . the thermal stability of horse - reddish peroxidase and asparaginase entrapped in alumina ( hrp @ alumina and asp @ alumina ) while acp was the subject of the detailed study , it was important to learn if the exceptional thermal stability induced by the alumina matrix was a general phenomenon which holds also for other enzymes and hrp and asp were selected for that purpose . in fig1 a , the activity of free hrp is compared to the activity hrp @ alumina after incubation for one hour at two temperatures , 37 and 75 ° c . whereas for the free hrp the drop in activity was a factor of 90 , to less than 1 . 2 % of the original activity , for the entrapped enzyme the reduction is only by a factor of 1 . 5 , to 65 % of the original activity . dsc analysis ( fig1 b ) showed that the denaturation temperature of hrp is shifted to a higher temperature by 30 ° c . when entrapped within the alumina matrix . as was the case with acp @ alumina , the cd spectra of the free and entrapped hrp are quite similar ( fig1 a ), indicating that the conformation of enzyme remains intact upon entrapment . the difference between the minima in the cd spectra — 205 and 210 nm for the free and entrapped enzyme is related to the change in environment from water to alumina . the free and entrapped enzyme were then heated from 20 to 90 ° c . at a rate of 30 c / min , and the ellipticity at the characteristic helical value of 205 nm was monitored ( fig1 b ). it is clearly evident that while the free enzyme underwent conformational changes above 65 ° c . ( the ellipticity drops ) in the case of the entrapped enzyme , the ellipticity remained constant in the interval 20 - 90 ° c . similar tests performed on asp @ alumina support the notion that entrapment in boehmite alumina provides major thermal stability . fig1 follows both the activity parameter and the cd - spectral parameter , and it is seen that the thermal stability observed for acp and for hrp is a feature of asp as well : the activity ( fig1 a ) of free asp the drop in activity upon heating by a factor of 51 , to less than 1 . 9 % of the original value , whereas for the entrapped enzyme the reduction is only by a factor of 1 . 4 , to 72 % of the original activity . the change in ellipticity due to heating the free and the entrapped asp shows the similar stability behavior as for hrp . the asp unfolding was monitored at 220 nm where the spectral changes were largest ( fig1 b ): it is seen that while free asp undergoes conformational changes sharply above 65 ° c ., the ellipticity of entrapped asp remains practically constant in the interval 20 - 90 ° c . the xylanase ( xlns ), proteinase ( prtns ) are traditional representatives of industrial enzymes . xylanase is the hydrolytic enzyme involved in depolymerization of xylan , finds application in many industrial processes such as enzymatic bleaching of paper pulp , juice clarification , extraction of oils from plants , texture improvement in bakery , bioconversion of agricultural wastes , bioscouring in textiles and improving digestibility of animal feed . proteinases ( prtns ) represent one of the three largest groups of industrial enzymes and find application in detergents , leather industry , food industry , pharmaceutical industry and bioremediation processes . acetylcholinesterase ( ache ) is used in a variety of chemical warfare applications , and is also a therapeutic enzyme . xlns , prtns ache were entrapped within alumina by adding the enzyme during the sol - gel manufacturing process . using kinetics , dsc and cd analyses , it is demonstrated that the alumina matrix not only protects form thermal denaturing but that over a certain temperatures range , it enhances quite significantly the activity of the enzymes . even extremely high temperature treatment (& gt ; 100 ° c .) does not lead to denaturation of entrapped protein . xlns @ alumina showed activity after the treatment at 105 ° c . during 10 min comparing with as - made sample , the treated remained 71 % (!) of initial activity . it has also been showed that the stability of entrapped enzymes was not limited by temperature , but also from the chemicals : after treatment with inhibitor of free ache the drop in activity was near of a factor of 3000 (! ), to less than 0 . 04 % of the original activity , for the entrapped enzyme we observed even minor increasing in the activity . for the entrapment of prtns , a mixture of 50 μl of glycine - naoh buffer solution ( ph 7 . 5 ) and 200 μl of freshly prepared alumina sol was transferred to a cuvette and then 20 μl of prtns ( 15 u / ml ) was added . ten minutes later the sol was left in vacuum desiccator at room temperature for 24 hours . the prtns @ alumina was rinsed inside final polystyrene cuvette with 1 . 0 ml of glycine solution ( ph 7 . 5 ) to assure removal of any adsorbed protein . the entrapment of xlns within alumina was carried out similarly with glycine - hcl buffer solution at ph 4 . 5 and 20 μl of xlns ( 30 u / ml ). the entrapment of all enzymes was full , as indicated by lack of activity in the supernatant solutions and washings . for the entrapment of ache , a 150 μl of freshly prepared alumina sol was transferred to a cuvette and then 50 μl of ache ( 500 u / mg ) was added . ten minutes later the sol was left in vacuum desiccator at room temperature for 24 hours . the ache @ alumina was rinsed inside final polystyrene cuvette with 2 . 0 ml of tdw to assure removal of any adsorbed protein . the entrapment of enzyme was full , as indicated by lack of activity in the supernatant solution and washing . prtns @ alumina . after rinsing with 1 . 0 ml of glycine solution ( ph 7 . 5 ), the bioactive hybrid was left for incubation at 37 ° c . for 30 min . then , the rinsing solution was replaced with 2 . 0 ml of 1 % weight / volume casein solution ( prepared by mixing 1 mg / ml of casein in the 50 mm potassium phosphate buffer ), 0 . 5 ml of folin &# 39 ; s reagent with following vortexing and the enzymatic activity was measured by following the formation of free tyrosine interacting with folin &# 39 ; s reagent by producing a blue colored chromophore measured as an absorbance value spectroscopically through the absorption at 660 nm , at temperatures ranging from 30 to 70 ° c . absorbance values generated by the activity of the enzyme are compared to a standard curve , which is generated by reacting known quantities of tyrosine with the folin &# 39 ; s reagent to correlate changes in absorbance with the amount of tyrosine in micromoles . the rinsing solution was also tested for enzymatic activity by the transfer of 2 . 0 ml of casein solution and 0 . 5 ml of folin &# 39 ; s reagent . for comparative analysis of free prtns , 2 μl ( 15 u / ml ) in 1 . 0 ml glycine solution ( ph 7 . 5 ) was taken and reacted similarly to the entrapped enzyme . xlns @ alumina . the enzymatic activities of free and entrapped xlns were determined using xylan as the substrate . the assay system was as a mixture of 2 . 0 ml of 1 % ( w / v ) xylan solution ( prepared by mixing 1 mg / ml of xylan in the glycine - hcl buffer solution at ph 4 . 5 ) and 0 . 1 ml of 0 . 04m dnsa solution . xylanase hydrolyzes the polymer xylan into the xylose monomers . the free xylose units produced as a result of xylanase activity react with dnsa reagent and form a colored complex that is measured by spectrophotometer at wavelength 540 nm . the enzymatic activity was measured spectrophotometrically as a rise in absorbance . absorbance values generated by the activity of the enzyme are compared to a standard curve , which is generated by reacting known quantities of xylose with the dnsa reagent . to polystyrene cuvettes containing xlns @ alumina in 1 . 0 ml glycine solution ( ph 4 . 5 ) was added , and comparative incubation was carried out at 37 ° c . for 1 h . the rinsing solution of xlns @ alumina sample was also tested for enzymatic activity by the transfer of 500 μl of the solutions into cuvettes and adding 2 . 0 ml of 1 % ( w / v ) xylan solution and 0 . 1 ml of 0 . 04m dnsa solution . the activity of xlns was determined by following the formation of reducing sugars . ache @ alumina . after rinsing with 2 . 0 ml of tdw , the bioactive hybrid was left for incubation at 37 ° c . for 30 min . then , the rinsing solution was replaced with 3 ml of tdw , 20 . 0 μl of 0 . 075m acetylthiocholine iodide and 100 μl of 0 . 01m dithiobisnitrobenzoic acid . the blank consisted of tdw , substrate , and dtnb solutions . the enzymatic activity was measured by following the formation of yellow anion spectroscopically through the absorption at 412 nm , at room temperature . the rinsing solution was also tested for enzymatic activity by the transfer of 1 ml of the solution into cuvette and adding 3 ml of tdw , 20 . 0 μl of the substrate and 100 μl of 0 . 01m dtnb . for comparative analysis of free ache , 5 μl ( 500 u / mg ) was taken and reacted similarly to the entrapped enzyme . for the test with inhibitor the aluminium chloride solution was taken . the 10 μl of 0 . 24m alcl3 solution was added to the cuvettes with free and entrapped ache before adding of substrate and kept during 5 min . testing the free enzymes . to compensate for the slower reactivity of the entrapped enzymes , lower concentrations of the free enzymes were taken —× 10 smaller all : xlns , prtns and ache enzymes . the obtained samples were characterized by ir spectroscopy on & lt ;& lt ; perkin elmer frontier ft - ir & gt ;& gt ;. specific surface areas , pore volumes and pore sizes distribution have been determined using the nitrogen adsorption - desorption method at 77 k ( micromeritics asap 2020 ). surface areas were calculated using the bet equation . pore volumes and pore size distributions were calculated using the bjh method . before the analysis the sample was degassed over night at room temperature . for scanning electron microscopy ( sem , ultrahigh resolution magellan 400 l electron microscope ), the final suspension of the entrapped enzyme was coated on silicon wafer and fully dried under vacuum . the spectral analysis of enzymatic activity was carried out using hp 8453 diode array spectrophotometer . dsc curves were obtained with 204 f1 phoenix netzsch apparatus and a heating rate of 10 ° c . min − 1 was used from 30 ° c . to 150 ° c . under nitrogen . cd spectra were recorded on a spectropolarimeter j - 810 ( jasco corporation ). the measurements of solutions were taken using a 2 - mm path length cuvette and coated slides were used for the entrapped proteins at 20 ° c . thermal stability studies were performed between 20 and 90 ° c ., at a constant heating rate of 3 ° c ./ min . individual components ( proteinase and alumina ) and also the composite prepared by entrapment of prtns within alumina matrix were studied by ftir analysis . the assignment of absorption bands in spectra was performed according to the literature data . the results clearly show a superposition of the typical absorptions bands of the matrix and the dopant . the main absorption bands of boehmite correspond to the following vibrations at the regions 400 - 830 cm − 1 and 1050 - 1070 cm − 1 related with al — o — h vibrations . a wide band in the region of 3000 - 3600 cm − 1 reveals the presence of non - equivalent water molecules , hydroxo groups and hydrogen bonds . however , the main difference of alumina and proteinase is the emergence of i and ii amide bands revealing the presence of the protein . the amide bands resulted from bending vibrations of the n — h / c — n bonds appears in the region of 1510 - 1600 cm − 1 . fig2 shows the morphology of the final prtns @ alumina composite , which are densely packed . similar morphology was obtained for xlns @ alumina . surface area and porosity analyses ( by nitrogen adsorption , analyzed by the bet and bjh equations ) confirmed with typical micro - mesoporosity ( fig2 ). for the composite prepared by entrapment of prtns — the values were : surface area of 174 m 2 / gr , pore volume of 0 . 116 cm 3 / gr and pore size of ˜ 3 . 5 nm . it needs to be assumed here that the protein release from alumina matrix strongly depends on proteins size and average pore size of matrix . in this case , maximum of proteinase release which was reached during 25 hours is only 7 %. similar behavior was observed for xylanase entrapped in alumina : there is a plato of 4 % release at 15 hours . in solution the typical thermal behavior of prtns is characterized by the expected gradual decrease of activity at temperatures above 40 ° c ., coming to a full stop of activity at temperatures exceeding 70 ° c . ( fig2 a ). similarly to the case of the theraputic enzymes , the stability of alumina - entrapped prtns was found to be so high that its activity increases with temperature up to 60 ° c . (!) ( fig2 b ). decrease in activity begins beyond that temperature . furthermore , another manifestation of the thermal stability of the entrapped prtns is its stability to prolonged cycle periods of heating to 60 ° c . and cooling ( fig2 d ). taking into account that proteinases are used in many hard and harsh areas of industry this fact is even more important for practical application . as the enzymatic decomposition of casein is the first - order , the temperature dependence of the rate constant is describable by arrhenius &# 39 ; law , k = a · e − e a / rt , ( where k is the rate constant , ea is the activation energy , t is the temperature , r is the gas constant and a is the arrhenius pre - factor ). the data in fig2 was thus subjected to an arrhenius analysis , and the results are shown in fig2 . the negative slope corresponds to a activation energy of the casein decomposition reaction of 74 kj / mol ; that is , up to 60 ° c . we have the classical increase of activity with temperature . then there is the positive slope with an apparent negative activation energy , which represents the temperature range where denaturation occurs , that is , the activity of the enzyme is disrupted by the heating . in solution the enzymatic reaction activation energy is lower — 43 kj / mol — and it is attributed to the higher value for the entrapped to diffusional restrictions of the big molecules of casein inside the mesoporous network , until the enzyme is reached . for many industrial applications , thermal stability of the enzyme is the more important feature . fig2 b shows that specific activity of the entrapped prtns and that it depends on the extent of doping of alumina by the enzyme . the decrease in specific activity as the enzyme concentration in the monolith increases can be caused by aggregation or denaturation of the enzyme in matrix . it may be also a consequence of a small decrease in accessibility of the enzyme to the substrate as the enzyme concentration increases . similar dependence of activity on concentration has been reported for encapsulated trypsin . to evaluate effect of aggregation of enzyme by increasing of loading within sol - gel matrix , we offer very simple explanation ( fig2 c ). taking account that available surface area of enzyme ( s ) is the function of composite activity ( a ) and volume of enzyme ( v ) is the function of concentration ( c ) we can conclude the following equation : according the classical equation s ˜ v⅔ . so , if the exponent in the fit correction is near to ⅔ ( 0 . 67 ) we deal with homogenous distribution of the dopant , where practically each of the enzyme unit take part in the reaction and aggregation is absent . if this value less than 0 . 67 aggregation occurs . in this case exponent factor is 0 . 59 , which means that aggregation takes place , but the value is quite close to ideal 0 . 67 where aggregation negligible . two additional measurements were employed to investigate the thermal stability . the first of these is comparative circular dichroism ( cd ) follow - up . since the cd spectrum is sensitive to conformational changes and is a common spectroscopic method for studying enzyme structure , one can use this tool for detection of denaturation as well . as observed from the cd spectra of the free and entrapped enzyme are quite similar , indicating the ( non - trivial result ) that the conformation of enzyme remains intact upon entrapment . the free and entrapped enzymes were then heated from 20 to 90 ° c . at a rate of 30 ° c ./ min , and the ellipticity at the characteristic helical value of 225 nm was monitored . it is clearly evident that while the free enzyme undergoes conformational changes above 30 ° c . ( the elipticity drops ) in the case of the entrapped enzyme , the ellipticity remains constant in the interval 20 - 90 ° c . while the free enzyme changes the cd spectrum due to total denaturation in the case of the entrapped enzyme , the ellipticity remains similarly to native form . yet another proof of the enhanced thermal stability comes from differential scanning calorimetry ( dsc ) analysis . the result of this analysis , which was developed for entrapped protein stability indicator , follows the changes in the denaturation temperature upon entrapment . dsc analysis ( fig2 ) shows that the denaturation temperature of prtns is shifted to a higher temperature by 23 ° c . when entrapped within the alumina matrix , which is in a good agreement with kinetic data . the thermal stability of xylanase entrapped in alumina ( xlns @ alumina ): while prtns was the subject of the detailed study , it was important to check if the exceptional thermal stability induced by the alumina matrix is a general phenomenon which holds also for other enzymes — industrial in this report — and xylanase was selected for that purpose for reasons described in the introduction . in fig2 a the relative activity of free xlns is compared to the activity xlns @ alumina at different temperatures in between 30 - 80 ° c . even stronger thermal stability was observed compared to prtns . whereas for the free xlns the activity drops after 40 ° c ., for the entrapped enzyme the relative activity increases up to extreme 80 ° c . (! ), where practically full list of existing enzymes loose the activity at all . dsc analysis ( fig2 b ) shows that the denaturation temperature of xlns is shifted to a higher temperature by 30 ° c . when entrapped within the alumina matrix . as was the case with prtns @ alumina , the cd spectra of the free and entrapped xlns are quite similar ( fig2 a ), indicating that the conformation of enzyme remains intact upon entrapment . the free and entrapped enzyme were then heated from 20 to 90 ° c . at a rate of 30 ° c ./ min , and the ellipticity at the characteristic helical value of 215 nm was monitored ( fig2 b ). it is clearly evident that while the free enzyme undergoes conformational changes above 50 ° c . ( the elipticity drops ) in the case of the entrapped enzyme , the ellipticity remains practically constant in the interval 20 - 90 ° c . thermal unfolding of the enzyme causes precipitation in solution , as we observed during experiment , but not in the alumina glass . both incomplete unfolding and lack of precipitation can be attributed to the influence of the alumina matrix . the enzyme molecules are relatively constrained by this matrix , which restricts the mobility of the protein molecules that is required for complete unfolding . because the enzyme molecules are isolated in separate “ cages ” in porous alumina , they cannot aggregate together and precipitate . the relatively small slope ( between 50 - 70 ° c .) of the plot may be an indication that the protein molecules exist in different microenvironments and have somewhat different stabilities toward unfolding . taking account the high thermal stability of xylanase entrapped within alumina the test of activity after sample incubation at 105 ° c . during 10 min was carried out . unexpectedly , the sample showed activity and even on a high level . comparing with as - made sample , the treated remained 71 % (!) of initial activity . fig2 a shows the activity of the free and entrapped enzyme as a function of the time . the activity of rinsing solution has been analyzed and was found to be negligible , similarly to the non - enzymatic hydrolysis . activity of 5 μl of free ache is much higher than for 50 μl entrapped . in fig2 b the activity of free ache is compared to the activity ache @ alumina after incubation with inhibitor . whereas for the free ache the drop in activity was near of a factor of 3000 (! ), to less than 0 . 04 % of the original activity , for the entrapped enzyme we observed even minor increasing in the activity . very high stability , but lower reaction rates . for mentioned application , stability of the enzyme and ability to work in presence of inhibitor are the more important features . for the entrapment of csp , a mixture of 0 . 4 ml of freshly prepared alumina sol was transferred to the quartz cuvette and then 50 μl of cisplatin 0 . 03m solution was added . ten minutes later the sol was left in vacuum desiccator at room temperature for 24 hours . the csp @ alumina was covered inside final quartz cuvette with 3 . 0 ml of histidine buffer solution ( ph 7 . 4 ) to measure the rate of drug release . the obtained samples were characterized by ir spectroscopy on & lt ;& lt ; avatar 360 fttir esp & gt ;& gt ;. specific surface areas , pore volumes and pore sizes distribution have been determined using the nitrogen adsorption - desorption method at 77 k ( quantachrome nova 1200 series e ). surface areas were calculated using the bet equation . pore volumes and pore size distributions were calculated using the bjh method . before the analysis the sample was degassed for 12 hours at 80 ° c . the crystal phase and crystallinity of the samples have been studied by x - ray diffraction method ( bruker d8 advance ) using cu - kα irradiation ( λ = 1 . 54 å ), samples being scanned along 2θ in the range of 4 - 75 ° at a speed of 0 . 5 degrees per minute . the spectral analysis of drug release was carried out using pg instruments t 70 + uv / vis spectrophotometer at the 203 nm with extinction coefficient 5 . 2 mm - 1 cm − 1 . transmission electron microscopy investigations was made using philips tecnai g2 f20 transmission electron microscope ( roanoke , va ., usa ) with an accelerating voltage of 200 kv . individual components ( cisplatin , alumina ) and also the composite prepared by entrapment of cisplatin within alumina matrix were studies by ftir analysis . the assignment of absorption bands in spectra was performed according to the literature data . the results clearly show a superposition of the typical absorptions bands of the matrix and the dopant . the main absorption bands of boehmite correspond to the following vibrations at the regions 400 - 830 cm − 1 and 1050 - 1070 cm − 1 related with al — o — h vibrations . a wide band in the region of 3000 - 3600 cm − 1 reveals the presence of non - equivalent water molecules , hydroxo groups and hydrogen bonds . the same absorbance can be found on the composite as well . however , the main difference of composite and boehmite is the appearance of amino band revealing the presence of the cisplatin . the absorption reveals at 1300 cm − 1 and is related with vibrations n — h band . next , it is important to determine the type of the alumina synthesized . of the six crystalline phases of alumina the biologically active form of alumina used as an adjuvant in current vaccines is boehmite / pseudoboehmite . xrd analysis of alumina indicates typical boehmite structure and scherer - equation analysis provides an elementary crystallite size of few nm ( typically 3 - 4 nm ). these results are in a good agreement with common alumina adjuvants which are also boehmite with crystallite size 4 - 5 nm . the sample presented a narrow pore size distribution by the bjh method . the sample exhibits typical microporous isotherm , according to the iupac classification , associated with the presence of micropores . the morphology of obtained samples was investigated by high resolution transmission electron microscopy . the boehmite sol - gel matrix consists of well - crystallized nanorods with average size 1 × 10 nm forming dense organized structure . the average pore size is about 2 - 3 nm , which is in a good agreement with nitrogen physisorption data . taking into account the global market of biomaterials with slow drug release and controlled delivery , the present work is of major applied importance . this brings to a highlight another important feature of the composites prepared by our special ultrasonic methodology : for targeted delivery of medicines , nanoparticles with the size of less than 100 nm are needed in order to overcome the biological barriers and in order to pass through capillaries . while the traditionally applied alumina - based adjuvants have size in the few microns range , the elementary building blocks of our alumina are around several tens of nm . the kinetics of csp release from the alumina into histidine buffer ( ph = 7 . 4 ) was measured and the results are presented in fig3 . it is seen that the profile of cisplatin release from alumina is represented by gradual release . this behavior was found to fit the weibull model for both cases here , m ( t ) is the dopant fraction that is released into the extracting solvent at time t , m ∞ is the total fraction that may be extracted by the chosen solvent after infinite time , tc is a characteristic time , namely the time required for 63 . 2 % ( exp (− 1 )) of the total extractable population be extracted , and b is a shape parameter , which may have values between 0 and 1 . if b = 1 , the model reduces to a simple first - order model ; if b & lt ; 1 , the curve is parabolic , with a higher initial slope ( accommodating the initial ‘ burst ’ observed in some extractions ). the parameter b can thus be taken as an indication of the degree of homogeneity of the extractable population : a value near to 1 implies a relatively homogeneous extractable population with tc corresponding to its first - order characteristic time , while a value far from 1 implies sample heterogeneity . the long release kinetics of csp was studied as a function of time during two weeks . after 12 days assay , the studying systems showed similar trends of release . during the period , alumina showed 88 % release of csp .
provided is a platform for entrapment within alumina sol - gel carriers of labile biologically active materials such as proteins , therapeutic enzymes , enzymes of industrial relevance , antigens , and small molecules for achieving successful and efficient protective storage , protection from harsh environmental conditions such as heat , ph and chemicals , delivery to site and subsequent treatment and / or vaccination against diseases against which the active agents are targeted .