Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/40—Viruses, e.g. bacteriophages

Definitions

• the present invention relates to the production of coated material containing, as a core, solid particles such as virus, microorganisms, proteins, protein aggregates, nucleic acids, chemical substances etc. which have inherent charged surface or which may develop their potential electrostatic attraction to opposite charged material with the aim of enhancing the stability and/or biological activity of the said core material.
• coated material of the present invention is coated baculovirus useful as a pesticidal agent.
• Encapsulation is a coating process by which a thin film of polymer, biopolymer, wax, resin, or metal substance is deposited onto a core to produce microcapsules.
• This kind of coating is used to protect the core material from its surroundings by means of a wall membrane, to control the time place or rate at which the core material (active agent) is released or even to label the said core material to obtain a complex product useful as diagnostic agents, pharmaceuticals, herbicides, pesticides, insecticides, etc.
• the coated particles often exhibit properties, which are significantly different to those of the template core, being attractive, both from a scientific and technological viewpoint.
• the mixture of gelatin and a suitable anionic polymer is diluted with warm water followed by the addition of an acidic aqueous solution such as acetic acid to reduce the pH of the system to the isoelectric point of gelatin or below, i.e. to values varying between 4.0 and 5.0 to allow chemical reaction between the polymer and gelatin to occur.
• an acidic aqueous solution such as acetic acid
• This process uses pH adjustment to provide hardness to the wall of coacervates.
• FR 2 675 398 which is related to sunlight protection microcapsules, also uses the physicochemical phenomenon called coacervation.
• the process comprises (i) preparation of a polymer colloidal solution and a dispersion of the substance to be encapsulated, (ii) a separation phase (coacervation) with formation of a three-phase system by varying pH value and, therefore, (iii) encapsulation of the dispersed substance.
• the pH values used in the process range from 3 to 7, preferably from 4 to 5. In this process, pH adjustment is used to permit the separation phase to recover coated particles (coacervates).
• EP 972563 describes a process for preparing coated capsules and hollow shells by coating particles with alternating layers of oppositely charged nanoparticles and polyelectrolytes. It is mentioned that the driving force for the multilayer film build-up is primarily due to electrostatic attraction and the formation of a complex among the charged species, which are deposited.
• the process comprises coating the template particles with alternating coatings of nanoparticles and polyelectolyte molecules which have ionically dissociable groups, e.g.: (i) polyacids, such as polyvinyl or polystyrene phosphoric acid, polyvinyl or polystyrene sulfuric acid, polyvinyl or polystyrene sulfonic acid, polyvinyl or polystyrene phosphonic acid, polyacrylic acid and their respective salts, or (ii) polybases, such as polyamines, or poly(ammonium salts).
• polyacids such as polyvinyl or polystyrene phosphoric acid, polyvinyl or polystyrene sulfuric acid, polyvinyl or polystyrene sulfonic acid, polyvinyl or polystyrene phosphonic acid, polyacrylic acid and their respective salts
• polybases such as polyamines, or poly(ammonium salts.
• the pH of the aqueous dispersion is adjusted in such a way that the molecules in each alternating layer, e.g. polyelectrolyte molecules and nanoparticles, each have opposite total charges.
• the formation of hollow shells represents an embodiment of particular importance for the use of the shells as a permeable wall.
• the permeability properties of the shell may be modified by selecting the conditions for decomposing the core, e.g. by selecting the temperature and heating conditions in a calcination procedure.
• the example is directed to the preparation of alternating SiO 2 -poly (diallyldimethylammonium chloride) (PDADMAC) multilayers and cites the fact that larger amounts of SiO 2 are adsorbed when the adsorbing solution contains NaCl and that the isoelectric point of the SiO 2 particles is 3, therefore SiO 2 is negatively charged under the conditions of adsorption (pH 5-6).
• PDADMAC diallyldimethylammonium chloride
• US 5,792,903 relates to the radioactive chitosan complex formed by labeling a chitosan, a biocompatible and biodegradable natural polymer, with radionuclide, a radioactive chitosan macroaggregate formed by making chitosan complex into particles, and a kit for preparing radioactive chitosan complex, process for preparation thereof and the use thereof for an internal radiation therapeutic agent.
• the process for preparing the internal radiation therapeutic composition of claim 1 comprising: (a) irradiating a water-soluble stable radionuclide compound with neutrons in a nuclear reactor to convert the water soluble stable radionuclide compound into an active radionuclide compound; (b) dissolving the active radionuclide compound in water to form a solution; (c) dissolving a chitosan in acidic solution (pH 2-4) to form a chitosan solution; and (d) adding the active radionuclide compound solution to the chitosan solution to form the internal radiation therapeutic composition.
• the preparation of the therapeutic composition is based on the solubility characteristics of a polymeric material (chitosan), in the good biocompatibility and biodegradability properties of chitosan and in the binding reaction, which occurs between the radionucleotide compound and chitosan. It must be emphasized that there is not a radionucleotide compound coating but a gel radioactive chitosan complex macroaggregate formation when the pH of the solution is adjusted to a nearly neutral value (physiological condition).
• US 5,965,123 relates to coated pesticidal agents which retain a significant amount of their original activity after exposure to ultraviolet radiation.
• the process comprises the steps of: (a) preparing an aqueous mixture of a pH-dependent polymer, (b) dissolving the pH- dependent polymer by adjusting the pH of the mixture of step (a) with a base to a pH above the solubilization pH of the pH-dependent polymer; (c) adding a pesticidal agent, an ultraviolet protector, optionally a stilbene compound, optionally a disintegrating agent and optionally a glidant to the solution of step (b) and blending to produce a homogeneous suspension containing dissolved pH-dependent polymer; (d) drying the homogeneous suspension of step (c); and optionally (e) milling the dried material of step (d).
• Pesticidal agents are inseticidal pathogens such as viral pathogens, bacterial pathogens and fungal pathogens.
• Niral pathogens are wild gypsy moth ⁇ PN, Autographa califomica ⁇ PN Douglas fir tossock moth ⁇ PV, European pine saw fly ⁇ PV and Helliothis zea ⁇ PN.
• pH-Dependent polymers are selected from the group consisting of methacrylic acid and methyl methacrylic copolymers, maleic anhydride and styrene copolymers.
• Claim 2 defines the pH adjustment made in step (b) as between 8.5 and 10.
• the object of the present invention is to provide enhanced particle coating by changing pH condition to modify the surface charge of the particle to be coated with the aim of minimizing or eliminating hindering forces that react against an efficient coating-template particles' binding.
• a first embodiment of the invention refers to stable coated particles comprising (a) a core consisting of a material which is inherently surface charged or which may develop its potential electrostatic attraction to opposite charged material and (b) a surrounding thin layer of a matrix comprising a polymer selected from the group consisting of polyvinyl or polystyrene phosphoric acid, polyvinyl or polystyrene sulfuric acid, polyvinyl or polystyrene sulfonic acid, polyvinyl or polystyrene phosphonic acid, polyacrylic acid and their respective salts and optionally ultrafine particles, wherein the core particles are irreversibly and individually coated.
• a second embodiment of the invention refers to durable coating baculovirus particles comprising (a) a core consisting of a virus particle selected from the group consisting of Baculovirus anticarsia and a polyhedrosis virus which is inherently surface charged and (b) a surrounding thin layer of a matrix comprising about 5 to 30% of a polymer selected from the group consisting of polyvinyl or polystyrene phosphoric acid, polyvinyl or polystyrene sulfuric acid, polyvinyl or polystyrene sulfonic acid, polyvinyl or polystyrene phosphonic acid, polyacrylic acid and their respective salts and optionally ultrafine particles, wherein the core particles are durable and individually coated.
• a third embodiment is a process for the preparation of coated particles comprising the steps of: (a) suspending a polymer selected from the group consisting of polyvinyl or polystyrene phosphoric acid, polyvinyl or polystyrene sulfuric acid, polyvinyl or polystyrene sulfonic acid, polyvinyl or polystyrene phosphonic acid, polyacrylic acid and their respective salts and optionally ultrafine particles in water to an appropriate concentration; (b) adjusting pH condition of the aqueous suspension of step (a) to a value lower than 4; (c) suspending the particles to be coated in water to an appropriate concentration and adjusting pH condition of the resulting suspension to a value lower than 4; (d) adding the suspension of step (b) to the suspension of step (c) and gentle stirring of the resulting mixture for a period of time enough to obtain a complete coating of the core particles; (e) adjusting the pH of the suspension of step (d) to 5-7 to obtain a neutral suspension of the
• a fourth embodiment of the invention refers to compositions containing the irreversibly coated Baculovirus, particularly the Baculovirus anticarsia, which is obtained according to the process described above.
• the physical form of these compositions can be granulates, tablets, dried powder or the like.
• Figure 1 illustrates the adsorption isotherm of baculovirus polyhedra coated with sulphate polystyrene latex particles having a particle size of 0.084 ⁇ m under pH 3.0; nads/n ⁇ v is the number of sulphate polystyrene latex absorved per baculovirus polyhedron; neq/ml is the number of sulphate polystyrene latex particles in equilibrium.
• Figure 2 exhibits the Scanning Electron Microscopy of baculovirus polyhedra coated with sulfate polystyrene latex particles having a particle size of 0.084 ⁇ m after neutralization with NaOH.
• Figure 3 shows the adsorption isotherm of baculovirus polyhedra coated with sulphate polystyrene latex particles having a particle size of 0.249 ⁇ m at (A) pH 3.0 and B) after neutralization; nads/nev is the number of sulphate polystyrene latex absorved per baculovirus polyhedron; neq/ml is the number of sulphate polystyrene latex particles in equilibrium; Figure 4 illustrates the Scanning Electron Microscopy of baculovirus polyhedra coated with sulphate polystyrene latex particles having a particle size of 0.249 ⁇ m at pH 3.0.
• Opposite charged colloidal particles and macromolecular interactions occur under different mechanisms such as coacervation and heterofloculation.
• the macromolecules frequently form a network which holds or keeps colloidal particles together as a core of the aggregate.
• the size and concentration relationships of the final aggregates are determinant on the final aggregates stability or particle precipitation as big aggregates.
• the predominance of one mechanism or another depends especially on the particle size of the species involved and on the existence of hindering forces against the core-coating binding.
• the coating of individual particles is favored and, uneven of coacervation, the geometry of particles is maintained in the heterofloculation process.
• the covering process is accomplished to enhance the coating- template particles' binding and consequently minimizing or eliminating these hindering forces with the purpose of obtaining a durable and sufficient covering while preserving the desirable properties of the template core.
• the modification of the surface charge of the particle to be coated by changing pH condition is determinant on neutralizing the aforementioned forces.
• the process is based on the pH lowering to less than 4, in which the surface charge changes from negative to positive and the hindering (mainly hydration) forces are neutralized.
• the polyhedra have an hydrophobic nature, first described by Small et. all (Small, D.A, Moore, ⁇ .F. and Entwistle, P.E. 1986.
• This invention provides small size aggregates containing particles individually coated by a fine and uniform layer constituted by molecules which have an average diameter 5-15 times smaller than the template particles.
• the particle size of the coating material is about 10 "3 to 1 ⁇ m.
• the core template is an organic, inorganic or biological solid material such as virus, microorganisms, proteins, protein aggregates, nucleic acids, chemical substances as those mentioned in US 4844896, etc.; having a specific structure and shape, e.g. polyhedrosis, spherical, rod-shaped. These template particles may develop their potential electrostatic attraction to opposite charged material or have inherent charged surface which can be changed by modifying the environmental conditions.
• the process of the present invention for preparing coated particles comprises the steps of:
• step (b) adjusting pH condition of the aqueous suspension of step (a) to a value lower than 4;
• step (d) adding the suspension of step (b) to the suspension of step (c) and gentle stirring of the resulting mixture for a period of time enough to obtain a complete coating of the particles;
• step (e) adjusting the pH of the suspension of step (d) to 5-7 to obtain a neutral suspension of the coated particles
• the Baculovirus' coating (BN particle size about 1,0 ⁇ m) is carried out under appropriate reagents concentrations to provide a ratio of 10 2 to 3 x 10 3 latex particles/polyhedrosis particle.
• Suspension of BN containing about 10 8 to 10 12 particles/ml and latex suspensions containing 10 n to 10 12 particles/ml are appropriated to obtain the above mentioned latex/virus ratio.
• the particle size of the latex material ranges from 10 "2 to 1.0 ⁇ m.
• the enhanced coating of the Baculovirus is based on the change of its surface charge from negative to positive at pH lower than 4, preferably at pH 3.0, in which the hydration forces are neutralized.
• ultrafine particles as an optional component of the coating layer, they can be selected from organic and inorganic particles, particularly inorganic particles, such as SiO 2 , TiO 2 , carbon black or alike.
• optical brightners may also be used to enhance the biological activity of the virus(Shapiro & Argauer, 1995).
• the coated particles may be recovered by using known purification and separation methods such as centrifugation, membrane processes, drying processes, etc.
• coated particles attained in the present invention have their stability and biological activity protected from their surroundings by means of the aforementioned fine and uniform coating which is made stable by neutralizing the hindering forces, e.g. hydration forces.
• the most preferred embodiment of the present invention is related to the covering of
• Baculovirus anticarsia (BV).
• the baculovirus is a double-stranded DNA occluded in a proteinaceos structure called polyhedron.
• This virus is an environmentally acceptable biological insecticide specific for control of velvetbean caterpillar Anticarsia gemmatatis, one of the main soybean defoliators in several countries.
• ultraviolet sunlight is the main destructive factor that affects the persistence of the virus in the field. Consequently, obtaining a good physical barrier against sunlight is desirable.
• the irreversible coating of this virus according to this invention means an excellent solution for this problem, decreasing or eliminating the amount of sunlight protectors in the commercial formulations which are frequently removed from the virus by the action of the weather conditions (rain, dew, etc).
• it is the guarantee of providing an efficient pesticide against Anticarsia gemmatalis.
• the coated polyhedrosis virus can be used in the field as an aqueous suspension. Most preferably, aiming to maximize its storage stability and facilitate its handling, the coated polyhedrosis is recovered from the suspension and dried to obtain a solid material which can be used as a dry powder or formulated as tablets or granulated mixtures.
• the coated polyhedrosis virus of the present invention can be formulated with known materials such as silica, attapulgite, kaolinite, bentonite, montmorilonite (see Medugno,CC, Ferraz, J.M.G, Maia, A.de H.N. & Freitas, C.CL. Evaluarion of a Wettable Powder Formulation for the nuclear Polyhedrosis Virus of Anticarsia gemmatalis (Lep.: Noctuidae). Pestic. Sci. 1997.51, 153-156).
• the larvae were ground, filtered through a synthetic fabric and diluted to a concentration of 10 g dm "3 of solids in 1% sodium dodecylsulfate (SDS). The suspension was then centrifuged at 5,000 xg and the solid resuspended in distilled and deionised water. The operation was then repeated until polyhedra of a high degree of purity could be observed by optical microscope; the concentrated dispersions were then kept at 4°C
• the suspension of Baculovirus (BN) to be coated was obtained by diluting the concentrated suspension to a concentration of 10 s to 10 9 BN particles/ml.
• Sulphate polystyrene latex stock solution commercially purchased, was diluted to the appropriate particle concentration (10 11 to 10 12 latex particles/ml). The pH of the suspension was adjusted to the final value of 3.0 with HC1 (analytical grade).
• the properties of sulfate polystyrene latex are given in Table 1. Table 1: Properties of sulfate polystyrene latex used to coat Baculovirus (from Certificate of Supplier) Size ( ⁇ m) Surface charge density Area per Charge ( ⁇ C/cm 2 ) Group (A 2 /C ( ⁇ H) ⁇ H 2
• EXAMPLE 3 Baculovirus coating with sulphate polystyrene latex having a particle size of 0.084 ⁇ m Heteroflocculation was achieved bv mixing the diluted sus ⁇ ension of Baculovirus. at a concentration of l,lxl0 10 particles/ml, as obtained according to Example 1, and the sulphate polystyrene latex suspension (particle size of 0,084 ⁇ m), as obtained in Example 2. The suspensions were mixed at 25°C and pH 3.0 under mild agitation in a centrifuge tube and to the proportion of 1200 latex particles per BN particle to obtain complete, fine and homogeneous coverage. The mixture was then centrifuged for 30 minutes at 10.000 rpm.
• the polyhedrosis core particle is positively charged and the sulphate polystyrene latex particles have a negative surface charge.
• the success of the coating process by heteroflocutation was determined by measuring the turbidity of supernatant.
• the number of particles not bound to Baculovirus was determined by turbidity measurements made on the supernatant at 400 nm and the absorbance value compared with that of the latex calibration curve measured at pH 3.0.
• Figure 1 shows the high affinity of particles.
• the ratio of added latex particles/polyhedrosis particles varied from 10 to 1600.1n order to verify the stability of the chemical reaction involved in the coating process, before centrifugation, the pH of a mixture sample was neutralized by adding 0.2 ml ⁇ aOH 0.1 ⁇ and absorbance was measured. After two cycles of centrifugation and washing with deionized water, the heterofloculated material was dried and the coating efficacy was documented by microphotographs obtained from a Scanning Electronic Microscope LEO estereoscan 440.
• Figure 2 illustrates the good results of the coating process of the present invention.
• Heterofloculation was performed as described in Example 3. The difference is related to the particle size of the coating material.
• sulfate polystyrene latex particle having a particle size of 0.249 ⁇ m. After this, the mixture was centrifuged at 2000 rpm.
• FIG. 4 shows a microphotograph obtained from a Scanning Electronic Microscope LEO estereoscan 440 illustrating these results.
• Heterofloculation was achieved by mixing, under gentle agitation, a fresh preparation of Baculovirus and a suspension of latex particles of 0.120 ⁇ m and 0.172 ⁇ m, positively charged with amidine (purchased from the Dynamic Corp.), in a concentration of 1200 particles/polyhedron at 25°C The mixture was then centrifuged for 30 minutes at 2000 rpm. Under this condition, latex do not precipitate and the number of particles not bound to BN can be determined by turbidity. The results evidenced the low affinity of the particles, showing coating factors of 64 and 56% to 0.120 and 0.172 ⁇ m particles respectively.

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• 2000
  • 2000-09-28 BR BRPI0015776-7A patent/BR0015776B1/pt not_active IP Right Cessation
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  • 2000-09-28 AU AU76351/00A patent/AU7635100A/en not_active Abandoned
  • 2000-09-28 JP JP2002529876A patent/JP2004509169A/ja active Pending
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