CN1040946C - Method and formulation for orally administering bioactive agents to and through the peyer's patch - Google Patents

Abstract

The present invention relates to a method and a prescription for an oral bioactive agent. The bioactive agent is prepared by that an active agent is encapsulated in excipient of one or more kinds of biodegradable biocompatible polymers or copolymers to form microcapsules. The microcapsule can pass through the gstrointestinal tract without influence and is absorbed by the Paiya's lymphoglandula.

Description

Be used to arrive the preparation method of the Orally administered composition of sending the Ya Shi lymph node

The present invention relates to be used to arrive the preparation method of the Orally administered composition of sending the Ya Shi lymph node.Bioactivator is by encapsulated in one or more biodegradable and biocompatible polymer or copolymer excipient.This capsule can make activating agent sending the Ya Shi lymph node and absorbed by this lymph node unaffected lymph node that arrives down the hair follicle shape that is animal.

Utilize micro encapsulation to prevent that responsive bioactivator degraded is well-known.Typically, bioactivator is by encapsulated in a kind of material of protecting film.The kind of this material is polymer normally.The method of encapsulated bioactivator can be to wrap the polymer masses of a monolayer outside activating agent, or activating agent is dispensed into equably in the model of a polymer.Activating agent dosage in the microcapsule can be complied with required change, and scope can be from trace to the composition that accounts for 95% microcapsule.The diameter of microcapsule also can be complied with required change, scope by less than 1 micron to greatly to 3 millimeters or more.

Sending the Ya Shi lymph node is the combinate form body of lymphatic nodule.It is positioned at ileum or intestines Lower Half, is the significant points that health defence alien bacteria infects.Antigen is the material that enhancing antibody forms, and it comprises extraneous protein or tissue.All antibody belong to immunoglobulins.When an antibody and antigen in conjunction with the time, they can become a kind of complex of non-activity, antigen has therefore neutralized.

Send the Ya Shi lymph node to have immunoglobulin A precursor B cell.These cells are grown and are born in gastrointestinal lamina propria district and upper respiratory tract, and can break up the synthetic property plasma cell of mature immunoglobulin A.These plasma cells are the secretory antibody molecule effectively.In the research of Heremans and Bazin, measure growing of the interior immunoglobulin A of oral antigen mice body, the result shows a series of unique antigen immune globulin A plasma cell and occurs.Appear at mesenteric mesaraic lymphatic nodule when just beginning, then appear at gland, appear at gastrointestinal lamina propria zone at last.(Bazin, H, Levi, G., and Doria, G., immunoglobulin A form the antibody cell for immunoreactive main effect, and this immunoreation is to measure in the outer gastrointestinal lymphoid tissue of oral contact antigen germfree mouse.J.Immunol.105:1049:1970 and Crabbe, P.A., Nash, D.R., Bazin, H.Eyssen, H, and Heremans, J.F. oral immunity or with the igA antibody in the intestinal plasma cell of the non-germfree mouse through the intestinal immunization of ferritin.J.Exp.Med.130：723；1969)。Therefore send the Ya Shi lymph node to it is apparent that the source of precursor immunoglobulin A cell, and it is being under being subjected to that the antigen body is responsive and stimulating, along circuitous movable passageway, the immunoglobulin A of release is in mucomembranous surface appearance at a distance.This round about manner provides a common mucomembranous immune system, promptly arrives mucosal areas by continuing to transport the B cell that is subjected to sensitization, with the antigen and the potential cause of disease of reaction intestines near zone.

Special significance of the present invention is the ability that is to induce with the oral immunity method protection antibody.Animal digests antigen, and causing unique antigen sIgA antibody to appear in bronchus or the nose liquid has been well-known.For example, studies show that what put into effect and test the effectiveness of oral influenza vaccine is that the secretion inducing influenza emits antibody in nasal discharge with the mankind.

Significantly, relate to any method or the prescription of Peroral active agent, all have the protection activating agent by gastrointestinal tract the time, to avoid the design of degrading.If do not pass through particular design, oral agents can be sent the Ya Shi lymph node to arrive under the situation of inappropriate amount or inefficacy.Under the form that is not protected, a large amount of bioactivators must be digested a specific effective dose earlier could arrive that to send Ya Shi lymph node, result be that most activating agent can not be absorbed.And, transport activating agent to sending the Ya Shi lymph node in order to increase, repeatedly the oral of number is necessary.Like this repeatedly counting oral high dosages of active agents not only wastes but also inconvenient.

Therefore, be necessary to find out an oral vaccine, the immune system of stimulating mucosal effectively, and can overcome bioactivator and arrive the problem of degrading when sending the Ya Shi lymph node passing gastrointestinal tract.More particularly need is to find out a method promptly to transport non-degradable antibody and arrive and send the Ya Shi lymph node.

The present invention is about utilizing oral way to transport the method for sending Ya Shi lymph node and the prescription of bioactivator to animal.Bioactivator in biodegradable and biocompatible polymer or copolymer by encapsulated.Capsule can make bioactivator not have degraded or only be subjected under the situation of small amount of degradation by gastrointestinal tract, and activating agent is not changed when sending the Ya Shi lymph node and the amount of remaining valid also arriving.The biocompatible definition of term is: a kind of polymer masses can not poisoned health, can be not carcinogenic and can not bring out the bodily tissue inflammation, this material should be that the biodegradable meaning is, can be degraded into the material that can be cleaned out easily by health by physiological process, and can not be accumulated in the health.This microcapsule also has simultaneously can be by the size of sending Ya Shi lymph node selectivity to absorb.Therefore, how allowing bioactivator arrive the problem of sending the Ya Shi lymph node and being absorbed has solved.

The method that the purpose of this invention is to provide the oral bioactivator of a kind of animal can arrive bioactivator to send the Ya Shi lymph node and is absorbed.Activating agent can not lose under the usefulness by animal gastrointestinal tract the stimulating mucosal immunity system.

Further object of the present invention provides a prescription, the center part and a kind of tool that comprise bioactivator are biodegradable and the bio-compatible characteristic and encapsulated in polymer or copolymer excipient, and they can be used with oral way as mentioned above.

Explain orally the embodiment of the invention method of carrying out.Just refer to, prolong mice is transported encapsulated antigen trinitrophenyl key hole  hemocyanin in 50: 50 poly-(the DL-lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether).

But should be noted that other polymer are also available except poly-(the DL-lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether).Below for example but be not limited thereto, as polyglycolic acid, the blended copolymer of DL-lactide and Acetic acid, hydroxy-, bimol. cyclic ester, copolymerization oxalates, polycaprolactone, poly-(lactide is caprolactone altogether), polyesteramide, poe and poly-beta-hydroxy-butanoic acid.

Also have, the other biological activating agent also can adopt.Below for example but be not limited to the antigen of lifting, as inoculate the antigen of anti-filterable virus, bacterial antigens, protozoon antigen, the antigen of fungal disease such as influenza collateral line virus, haemophilus antigen, coccobacillus pertussis antigen, eisseria antigen, drench Proantigen, streptococcus pneumoniae antigen, the antigen that falciform malaria worm antigen or pathogenic microorganism induce an illness, or inoculation antagonism by microorganism such as anthelmintic cause of disease diseases induced antibody.

I. microcapsule

(A) for sending the Ya Shi lymph node to penetrate research preparation dyestuff filling microcapsule

Coumarin, a water-insoluble dyestuff is with polystyrene and a kind of non-biodegradation polymer micro encapsulation.Purpose provides and can be used for following microcapsule and penetrate the colored microcapsule of sending the Ya Shi lymph node.The method for preparing this microcapsule is as follows: the first, prepare a kind of polymer solution, and method is polystyrene (the model 685d with 4.95 grams, Dow ChemicalCompany, Mildand MI) is dissolved in 29.5 gram dichloromethane (Reagent Grade, EastmanKodak., Rochester NY).(Warrington PA) adds in the polymer solution that mixes up for Polysciences, Inc., and makes the coumarin dissolving with a magnetic stirring rod stirring with 0.05 coumarin that restrains then.

In another container, prepare polyvinyl alcohol (PVA) aqueous solution of 10% (weight).The method for preparing this machining medium is that (Allenton PA) is dissolved in the deionized water of 360 grams for VINOL 205c, Air Products andChemicals with the PVA of 40 grams.In the PVA solution for preparing, it is saturated to add 6 gram dichloromethane solutions.Then PVA solution is poured into a 1L resin kettle that is equipped with a truebore shaft and one 2.5 inches polytetrafluoroethylene turbine propulsion devices (Ace Glass, Inc., Vineland, NJ) reuse Fisher stedi speed motor changes with per minute 380 and stirs.

Then, polystyrene/coumarin mixture is added the resin kettle that contains the PVA machining medium.This method is to utilize its 7mm internal diameter long neck funnel to guide mixture to enter resin kettle.Produce stable O/w emulsion, under atmospheric pressure stir about 30 minutes is to produce the oily microdroplet of suitable size.Then seal resin kettle, ease down to 520mm Hg gradually with resin kettle is intrinsic pressure with a water aspirator that is connected to barometer and discharging valve.Material stir about 24 hours under reduced pressure makes all dichloromethane evaporations in the pot.After the evaporation of all dichloromethane, the hardened microcapsule of centrifugal collection, and the vacuum chamber inner drying of room temperature 72 hours.

(B) preparation has antigenic microcapsule

With trinitrophenyl key hole  hemocyanin (TNP-KLH), water-soluble antigen encapsulated a kind of can be biocompatible, in the biodegradable polyester poly-(the DL-lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether).The program of preparation microcapsule is as follows:

At first 50: 50 poly-(the DL-lactide is total to Acetic acid, hydroxy-, bimol. cyclic esters) of 0.5 gram are dissolved in 4.0 dichloromethane that restrain and prepare polymer solution.Then with 300 microlitre TNP-KLH with aqueous solution (46mg TNP-KLH/ml; After decomposition), in that homodisperse is gone into is poly-(the DL-lactide the is Acetic acid, hydroxy-, bimol. cyclic ester altogether) solution.Must use Vortex-Genie 2 (Scientific Industries, Inc., Bohemia, NY) rotation mixture between dispersed phase.

In another container, 4.8 gram PVA are dissolved in the PVA aqueous solution of preparation 8% in the 55.2 gram deionized waters.After the PVA dissolving, with its add a 100mL resin kettle (KontesGlass, Inc, Vineland, NJ).This pot has a trubebore agitator and 1.5 inches politef turbine impellers.The polymer solution that mixes up is poured in the resin kettle that contains PVA preparation medium via a 7mm internal diameter funnel.Pour in the process, must stir PVA solution with per minute 650 rotations.Behind about 10 minutes of the O/w emulsion that in agitated kettle, forms, with the substance transfer in the resin kettle to the 4L beaker that the 3.5L deionized water wherein is housed.2 inches rustless steel impellers of reuse stir with per minute 800 rotation numbers, about 30 minutes of the microcapsule that forms in the agitating deionized water, and centrifugal collection, reuse deionized water rinsing 2 times is to remove the PVA of any remnants, and the reuse freeze-drying is collected.This microcapsule is made up of about 1 to the 10 micron spheroidal particle of diameter.

The method that mensuration has TNP-KLH composition in the antigen microcapsule (be microcapsule in filling material) is as follows: scale goes out 10mg and has antigenic microcapsule in the 12mL centrifuge tube.Add the 3.0mL dichloromethane in pipe, and vortex moving poly-to dissolve (the DL-lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether), and then add in pipe and tempestuously moving 1 minute of vortex of 3.0mL deionized water, the composition in the centrifugalize centrifuge tube is told organic layer and water layer.

Water layer is transferred to a 10mL volume quantitative flask, again deionized water is added flask up to the mark place.Quantity and TNP-KLH the quantity microcapsule in of TNP-KLH in flask, the available protein calibrating is calculated.Calculate according to weight, microcapsule contains 0.2%TNP-KLH.

II. biological research

(A) mice

BALB/C mice in 8 to 12 ages in week, is used in this research.

(B) trinitrophenyl key hole  hemocyanin

Key hole  Megathura Crenulata hemocyanin (KLH) purchase in Calbio chem (San Diego, CA).Program (Rittenburg, M.B. and Amkraut, the immunity of A.A. trinitrophenyl hemocyanin: produce primary and secondary antihapten precipitin, J.Immunol.97:421 according to Rittenburg and Amkraut; 1966).Replacement rate is that spectrophotometry is TNP861-KLH, this is to use 15400 ear extinction coefficient not under the 350nm wavelength, and to the release of the KLH under this wavelength employing 30% correction (Rittenburg, M.B. and Amkraut, A.A. the immunity of trinitrophenyl hemocyanin: produce primary and secondary antihapten precipitin, J.Immunol.97:421; 1966).

(C) immunity

Microcapsule and non-microcapsule TNP-KLH are suspended in tap water and 2 parts of sodium bicarbonate (7.5% solution) of 8 parts of filtration sterilizations with 10 μ g/mL antigen concentrations.The mouse fasting a whole night that to be tried earlier, pour into the suspension (Babb of 0.5mL again via the stomach intubation with cannula needle, J.L., Kiyono, H, Michalek, S.M. and McGhee, J.R.LPS immunoreation rule: suppress the immunoreation of oral T-dependence antigen, J.Immunol.127:1052; 1981).

(D) collection of biological fluid

1. serum

Behind puncture posterior orbit clump, collect blood with the capillary pipette that the nuclear school is used.After clot forms, concentrate serum and remove erythrocyte and platelet, make to lose vigor, and be kept at-70 ℃ with heat when calibrating with centrifuge.

2. intestinal secretion thing

Mice irritated 4 (0.5mL) irrigating solution (25mM sodium chloride at per 15 minutes interval, 40mM sodium sulfate, 10mM potassium chloride, 20mM sodium bicarbonate and 48.5mM polyethylene glycol, 530mosM permeability) (Elson, C.O., Ealding, W.and Lefkowitz, J., a kind of lavation technology allows repeated measure igA antibody in mouse intestinal secretion thing, J.Immunol.Meth.67:101; 1984).After pouring into 15 minutes of last drip irrigation washing liquid, mouse is anaesthetized.After 15 minutes intramuscular injection 0.1mg pilosines.Injection back 10 to 20 minutes stimulates the intestinal inclusions to drain.This excreta collection is being equipped with the accompanying in the Ti Shi ware of 3mL solution, and (Sigma, st Louis is MO) at the solution of 50mM EDTA to contain the 0.1mg/mL soybean trypsin inhibitor in this ware.Rotation accompanies the centrifugalize of Ti Shi ware to shift out float tempestuously, float is transferred in the poly-carbonic acid centrifuge tube of a round bottom.Before employing high speed centrifugation precipitation is made clarification, and necessary adding 30 μ L phenylmethyl sulfonylfluorides (PMSF, Sigma).After the clarification, add 20 μ L phenylmethyl sulfonylfluorides and 20 μ L1% sodium azides again, make in hyclone (FCS), to form 10% solution so that the substitute of keeping the protein enzyme to any to be provided.

3. saliva

With intestinal is excremental a large amount of salivations arranged simultaneously.With capillarity with the 0.25mL saliva collection to pasteur pipet, before clarification, must add soybean trypsin inhibitor, phenylmethyl sulfonylfluoride, Hydrazoic acid,sodium salt and FCS, every kind of 20 μ L respectively.

(E) immuno-chemical reagent

Be specific to Mus IgM, the polyclone sheep IgG antibody of solid-state absorption of IgG and IgA and protein affinity purification be commercially available (Southern Biotechnology Associates, Birmingham, AL).Appropriate according to them in conjunction with monoclonal antibody and the proteinic ability of bone marrow cell carcinoma, can measure their uniqueness at radioimmunoassay.

(F) solid phase radioimmunoassay

Use the toluene-sodium-sulfonchloramide method that antibody purification is put on carrier-free Na ¹²⁵I (Amersham).(Hunter, W.M. radioimmunoassay In:Handbook ofExperimental Immunology, M.Weir (editor).Blackwell?ScientificPublishing，Oxford.p.14.1；1978)。(Dynatech) is coated with TNP (trinitrobenzene) on Immulon Removawell mensuration bar, and this trinitrobenzene spends the night in BBS with bovine serum albumin (BSA) and combines with every mL1 μ g ratio under 4 ℃.Control stripes is not coated anything, but all strips were all at room temperature isolated 2 hours with 1%BSA in BBS.BSA is used for all samples and labelling ¹²⁵The diluent of I reagent.Biological sample liquid is diluted to contains 1 to 1000ng/mL special-shaped unique antigenic antibody, again the biological fluid sample is added 3 times and come and go in the pipes of washing, at room temperature cultivated then 6 hours, after washing with 1,000, the 000cpm labelling ¹²⁵The anti-immunoglobulin that I is unique special-shaped adds in each pipe, and in 4 ℃ overnight incubation.Washing is removed unconjugated ¹²⁵Behind the I antibody, pipe in the spectrometer of γ-5500, count (Beckman Instruments, Inc., San Ramon, CA).(Miles Scientific, Naperville IL) are placed on and are covered with 1 μ g/ and manage on the pipe of unique haterotypic antibody and calibrate to use two times of serial dilution standard serums.This standard serum contains known immunoglobulin quantity.It is to obtain by computer that calibration curve and unknown benefit are gone into, and computer program is to adopt " Logit-log " or " FourParameter Logistic " program in BASIC (RIA001 and RIA004).This program in Biomedical Computing Technology Center, have (Vanderbilt MedicalCenter, Nashville, TN).

(G) result

1. the dyestuff of preparation filling microcapsule penetrates and sends the Ya Shi lymph node

The size restriction that the investigation microcapsule enters the quantity of the lymphoreticular tissue relevant with intestinal and is subjected to when penetrating is to allow the oral polystyrene microcapsule that has the fluorescent dye coumarin of mice.In the BALB/C mice that does not have anesthesia and disconnected surfeit,, send the 100mg/mL suspension of 0.5mL to and go in the stomach of rats with a feeding pin.This suspension is that fluorescent capsule (diameter is less than 5 μ m or 8 to the 50 μ m) bubble of size of all kinds is made in tap water.Mouse can die at the different time of taking (after 0.5,1 and 2 hour), then with small bowel resection and isolate 1 centimetre of intestines that contain the scattered Ya Shi of group lymph node.Flushing lumen part is routed up and quick freezing.

Prepare freezing part, be absorbed into microcapsule number, place and the size of sending the Ya Shi lymph node by the enteric cavity pipe with the luminescence microscope examination.Though the microcapsule folder is trapped in the fine hair, has avoided capsule to be rinsed, and does not all see the phenomenon that penetrates tissue except sending the Ya Shi outside lymph node at any point.After oral half an hour, observe microcapsule and send the Ya Shi lymph node, but not distally at nearside.When time lengthening, microcapsule can be transported by peristaltic action, in 2 hours they by gastrointestinal tract, and appear at ileum send the Ya Shi lymph node.Microcapsule mainly is the existence of perimetric pattern.Away from sending Ya Shi lymph node dome center.This situation is given a kind of impression of people, and folder is trapped in dome and is close to lint and helped capsule to be absorbed when wriggling exactly.Reach the particle of 10 μ m though in sending the Ya Shi lymph node, seen some diameters, be absorbed the little glue Nang of maximum diameters, and in during examination, they are the capsule of the most deep Ya Shi of the group lymph node of finding still less than 5 μ m.This presentation of results, diameter are absorbed into by the enteric cavity pipe apace and optionally at the microcapsule of 1 to 5 μ m sends the Ya Shi lymph node.It is that effective ways are to transport the immunity that antigen brings out mucomembranous surface to the relevant lymphoid tissue of intestinal that the microcapsule that this suggestion is made up of the biodegradation membrane substance can be used as.

2. make oral vaccine with the biodegradable microcapsule of band antigen

Do peripheral material manufacturing and contain hapten proteantigen (TNP-KLH) with poly-(the DL-lactide is Acetic acid, hydroxy-, bimol. cyclic ester altogether) copolymer.According to size microcapsule and the capsule of diameter in 1 to 5 mu m range elected assess separately.These microscopic capsules contain 0.2% antigen according to the weight meter.Capsule can become effective antigen and transport system by digestion the time, its mode is that the 10mg/mL suspension of 0.5mL (10 μ g antigen) was cultivated 4 days through stomach in the aseptic tap water of sodium bicarbonate buffer.In order to make comparisons, another group mice employing oral immunity method too obeys into that 0.5mL does not have capsular TNP-KLH solution (20 μ g/mL).The only oral dilution of control group mice group.

The 14th day and the 28th day of immunity the last time, serum, saliva and intestinal secretion thing obtain from 5 jejunitas mouse of each group.These samples are measured all IgMs of sum of series of unique TNP, the antibody of the alloantigen of immunoglobulin G and immunoglobulin A (seeing Table 1) with unique special-shaped radioimmunoassay method.It nearly all is the immunoglobulin A level that saliva and intestinal secretion matter sample contain antigen.This result is consistent with research in the past and produce evidence, and proves the pollution that does not cause serum in the program that is used for collecting juice.Cause all immunoglobulins at different levels in institute's test liquid obvious change being arranged at present without any a kind of immunity record.Trace but the spontaneous generation IgM that can measure and IgG heterogeneous be anti--TNP antibody, and detected and is existed in the serum, the IgA heterogeneous in the serum and being subjected in the intestinal secretion thing of immune matched group mouse.Yet surpassing continuously, the 30 μ g micro encapsulation TNP-KLH that use same dose can cause all 14th day (see Table one in last hurdle) of heterogeneous after immunity in secretions that unique antigen I gA antibody appears at control group mice and the serum in 3 days.The 28th day antibody can increase higher after immunity.Opposite oral equal number does not have encapsulated antigen, in the liquid which kind of was test in office, does not have and induces the unique antibody of any abnormal shape to render a service.

(H) significance

This result of study merits attention in several respects.The first, significant unique antigen I gA antibody is induced in present serum and the mucosa secretions.Rarely or at all do not have in this immunization that is reflected at general use.Therefore this immunization method should impel immunity to strengthen significantly at mucosa, anus porch or in the pathological area at some antibacterials and virus causing disease.The second, the antigenic preparation of micro encapsulation be a kind of when oral effective immunogen; Working as oral equal number does not on the contrary have capsule antigen body, then lacks effective immunogen.Therefore, the enhancement effectiveness that microcapsule can be surprising, this point can be inferred by sending the Ya Shi lymph node to increase the amount that absorbs.The 3rd, immunoreactive induction period is seemingly competent.When not having excipient, the feature of proteantigen systemic immunity method is to be the antibody peak in 7 days to 14 days; And the oral antigen microcapsule that contains, the reaction of being brought out in the 28th day is higher than the 14th day.Therefore the biological corrosion of this explanation wall film material and antigen also bring out more competent reaction in discharging and betiding between the extended period.Table 1 brings out in the serum and mucosa secretions that unique antibody appears at mice with encapsulated oral immunity method

Immunoglobulin mg/ml sample

The sample of IgM immunoglobulin G immunoglobulin A immunogen immune artifact

Total resisting-TNP is total to be resisted-and TNP is total to be resisted-TNP

14 days intestinal secretion thing＜1＜1 62＜1 79 of time matched group, 355 25

Saliva＜40＜40＜40＜10 2,651＜10

Serum 445,11 65,503,726 37 1,470,553 32 non-encapsulated 14 days intestinal secretion thing 41 131＜1 64,985 17TNP-KLH saliva＜40 10＜40＜10 1,354＜10

Serum 298,733 11 6,000,203 29 1,321,14 days intestinal secretion thing 3＜1 130＜1 95 of 986 21 micro encapsulation, 368 222TNP-KLH saliva＜40＜10＜40＜10 1,461 88

Serum 360,987 1,461 5,788,813 572 1,411,312 1,077 non-encapsulated 28 days intestinal secretion thing＜1＜1 94＜1 88,661 64TNP-KLH saliva＜40＜10＜40＜10 1,278＜10

Serum 301,223 21 5,788,813 67 1,375,28 days intestinal secretion thing 4＜1 122 2 82 of 332 63 Wei Rubber capsuleizations, 869 422TNP-KLH saliva＜40＜10＜40＜10 1,628 130

Serum 320,192 1,904 5,901,503 2,219 1,277,505 1,198

Claims (9)

1. make oral and the bioactivator of induce immune response can be released into the method for compositions of sending the Ya Shi lymph node of described animal for one kind for animal, this method comprises: the bioactivator of effective dose is encapsulated in a kind of biodegradable biocompatible polymer or copolymer, thereby forming diameter is the microcapsule of 1 μ m-10 μ m, and wherein said microcapsule can be sent Ya Shi lymph node selectivity to absorb and can not degraded by gastrointestinal tract.

2. by the described method of claim 1, wherein said excipient is a polymer.

3. by the described method of claim 2, wherein said excipient is a co-polymer.

4. by the described method of claim 1, wherein said excipient is selected from polyglycolic acid, DL-lactide and Acetic acid, hydroxy-, bimol. cyclic ester mixed copolymer, copolymerized oxalate, polycaprolactone, poly-(lactide-be total to-caprolactone), polyesteramide, poe and poly-beta-hydroxy-butanoic acid.

5. by the described method of claim 1, wherein said excipient is poly-(DL-lactide-co-glycolide).

6. by the described method of claim 1, wherein said bioactivator is protozoacide antigen, virus antigen, fungal antigen or bacterial antigens.

7. by the described method of claim 6, wherein said antigen is trinitrophenyl key hole  hemocyanin.

8. by the described method of claim 7, the diameter of wherein said microcapsule is between 1 to 5 μ m, and described bioactivator is a trinitrophenyl key hole  hemocyanin, and described excipient is 50: 50 poly-(the DL-lactide is an Acetic acid, hydroxy-, bimol. cyclic ester altogether).

9. by the described method of claim 1, wherein said diameter is about 1 μ m to 5 μ m.