RO128007A2

RO128007A2 - Process for obtaining micro/nano-structured functional assemblies of chitosan and anti-inflammatory active substances by simultaneous self-assembling

Info

Publication number: RO128007A2
Application number: ROA201100525A
Authority: RO
Inventors: Simina Virginia Dreve; Liliana Olenic; Ioan Bratu; Irina Kacso; Maria Crişan
Original assignee: Institutul Naţional De Cercetare-Dezvoltare Pentru Tehnologii Izotopice Şi Moleculare
Priority date: 2011-06-01
Filing date: 2011-06-01
Publication date: 2012-12-28

Abstract

The invention relates to a process for obtaining liquid micro/nano-structured functional assemblies of chitosan and anti-inflammatory active substances by simultaneous assembling, by incorporating active substances with anti-inflammatory properties by complexing and/or physical adsorption, resulting in new products with controlled release of active substance comprising the ultrasound preparation, at 35 Hz, of chitosan hydrogels additivated with 2% by volume Tween-80 and 0.2% by volume oleic acid, at the ambient temperature, said chitosan hydrogels comprising previously admixed anti-inflammatory active substances, which, by simultaneous self-assembling permit film-type and cellular-type solid materials to be formed.

Description

PROCEDEU DE OBȚINERE A ANSAMBLELOR FUNCȚIONALE MICRO/NANO-STRUCTURATE DE CHITOSAN SI SUBSTANȚE ACTIVE ANTIINFLAMATOARE PRIN AUTOASAMBLARE SIMULTANAPROCESS FOR OBTAINING MICRO / NANO-STRUCTURED FUNCTIONAL ASSEMBLIES OF CHITOSAN AND ANTI-INFLAMMATIVE ACTIVE SUBSTANCES BY SIMULTANEOUS SELF-ASSEMBLY

Invenția se refera la un procedeu de obținere a unor ansamble funcționale micronice si submicronice de chitosan cu substanțe active antiinflamatoare, in scopul efectuării de studii teoretice si practice cu scop științific si a utilizării lor in medicina si farmacie. Ansamblele de chitosan cu substanțe active antiinflamatoare prezintă structuri supramoleculare formate prin încapsulare si se folosesc la cedarea controlata de pricipii bioactive [1,2] .The invention relates to a process for obtaining micron and submicron functional assemblies of chitosan with anti-inflammatory active substances, in order to perform theoretical and practical studies for scientific purposes and their use in medicine and pharmacy. Chitosan assemblies with anti-inflammatory active substances have supramolecular structures formed by encapsulation and are used in the controlled release of bioactive principles [1,2].

In societatea contemporana se pune tot mai mult accentul pe creșterea calitatii vieții. In acest sens exista preocupări majore pentru imbunatatirea calitatii medicamentelor urmarinduse ca medicamentele produse sa fie cit mai eficiente, sa aiba cit mai puține efecte secundare si sa fie cit mai complet caracterizate. Acțiunea medicamentelor si proprietățile fizico-chimice depind esențial de structura moleculara si cristalina a acestora. Cercetarea fundamentala este axata pe obținerea si caracterizarea fizico-chimica si structurala a ansamblelor supramoleculare de tip oaspete gazda. In literatura științifica se releva faptul ca au fost investigate substanțe bioactive de tip antiinflamatoare non steroidice, antidepresive, hepatoprotectoare, etc., numărul de lucrări științifice publicate fiind in continua creștere [3, 4,In contemporary society, more and more emphasis is placed on increasing the quality of life. In this sense, there are major concerns for improving the quality of drugs, aiming to make the drugs produced as effective as possible, to have as few side effects as possible and to be as fully characterized as possible. The action of drugs and their physico-chemical properties depend essentially on their molecular and crystalline structure. The fundamental research is focused on obtaining and physico-chemical and structural characterization of the supramolecular assemblies of host guest type. In the scientific literature it is revealed that bioactive substances such as non-steroidal anti-inflammatory drugs, antidepressants, hepatoprotectives, etc. have been investigated, the number of published scientific papers being constantly increasing [3, 4,

5]·5]·

Chitosanul este un biopolimer cu lanț de polizaharida, derivat din chitina deacetilata a crustaceelor cu proprietăți imunoadjuvante, hemostatice, epitelizante, antiseptice, etc. Utilizarea biomaterialelor derivate de chitosan ca baza pentru eliberarea controlata de substanțe bioactive este unul dintre subiectele de mare noutate in cercetarea in domeniu, mai ales datorita faptului ca penetrarea substanțelor active la nivel membranar este limitata de criteriul de dimensionalitate. Celulele umane sunt delimitate de membrane celulare, care protejează mediul intra-membranar, dar si permit penetrarea sau eliminarea de substanțe prin porii membranei, care au dimensiuni medii de 70 nm, in timp ce dimensiunea medie a celulelor este de cea. 10 pm. Preparatele de tip hidrogel de chitosan microstructurat pot fi administrate si intravenos, întrucât capilarele au dimensiuni de maxim 4 pm [6].Chitosan is a biopolymer with a polysaccharide chain, derived from the acetylated chitin of crustaceans with immunoadjuvant, hemostatic, epithelializing, antiseptic, etc. properties. The use of chitosan-derived biomaterials as a basis for the controlled release of bioactive substances is one of the topics of great novelty in research in the field, especially due to the fact that the penetration of active substances at the membrane level is limited by the dimensionality criterion. Human cells are delimited by cell membranes, which protect the intra-membrane environment, but also allow the penetration or elimination of substances through the pores of the membrane, which have average dimensions of 70 nm, while the average size of the cells is that. 10 p.m. Microstructured chitosan hydrogel preparations can also be administered intravenously, as the capillaries have a maximum size of 4 pm [6].

Prin realizarea de preparate micro-sau nanostructurate, cu dimensiuni mai mici sau egale cu 100 nm, avand matricea purtătoare a substanței active antiinflamatoare chitosanul, acesta asigura eliberarea si penetrarea treptata a subtantei active prin porii membranelor celulare [7], Particulele de aceste dimensiuni au proprietăți specifice, coerente si diferite de ale materialului similar ne-nanostructurat si dau multifunctionalitatea materialelor nanostructurate.By making micro- or nanostructured preparations, with dimensions less than or equal to 100 nm, having the matrix carrying the active anti-inflammatory substance chitosan, it ensures the release and gradual penetration of the active substance through the pores of cell membranes [7], Particles of these sizes have specific, coherent and different properties of similar non-nanostructured material and give the multifunctionality of nanostructured materials.

La nivel internațional exista preocupări continue pentru identificarea de noi compuși biocompatibili cu activitate tintita, si de noi terapii non-invazive, iar din acest punct de vedere produsele pe baza de chitosan castiga teren constant.Internationally, there are ongoing concerns for the identification of new biocompatible compounds with targeted activity, and new non-invasive therapies, and from this point of view chitosan-based products are gaining ground.

iOFICIUL OE STAÎ PENTRU INVENȚII Și MĂRCI Cerere de brevet de invenție cerere o e brevet de invențieOE OFFICE STAFF FOR INVENTIONS AND TRADEMARKS Patent Application Patent Application

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^-2011-00525-ο I -06In tara noastra produsele farmaceutice pe baza de chitosan au pătruns foarte timid, iar cercetările in domeniu sunt inca in faza de debut. In prezent exista pe piața medicamentelor o serie de produse de uz curent, cum ar fi: Brexin, Cicladol, Nitropen, etc, care utilizează ca vehicule moleculare in transportul tintit al diferitelor substanțe bioactive, matrici din categoria polizaharidelor de tipul ciclodextrinelor.^ -2011-00525-ο I -06In our country, chitosan-based pharmaceuticals have entered very timidly, and research in the field is still in its infancy. There are currently on the drug market a number of commonly used products, such as: Brexin, Cycladol, Nitropen, etc., which use as molecular vehicles in the targeted transport of various bioactive substances, matrices in the category of polysaccharides such as cyclodextrins.

In scopul realizării de ansamble micronice si submicronice de chitosan cu substanțe active antiinflamatoare au fost folosite pe plan internațional doua procedee: a) copolimerizarea sau autoasocierea simultana a chitosanului cu principiul activ de interes; b) prepararea nano- sau micro-capsulelor de chitosan urmata de adsorbtia sau legarea chimica prin tehnici specifice a substanțelor bioactive medicamentoase de livrat. Exista cateva lucrări de baza publicate recent in acest domeniu [8, 9]. Trebuie specificat ca in literatura nu s-a semnalat inca existenta unor sisteme ideale pentru eliberare controlata si tintita de medicamente.In order to make micron and submicron chitosan assemblies with anti-inflammatory active substances, two procedures were used internationally: a) simultaneous copolymerization or self-association of chitosan with the active principle of interest; b) preparation of chitosan nano- or micro-capsules followed by adsorption or chemical binding by specific techniques of the bioactive medicinal substances to be delivered. There are several basic papers recently published in this field [8, 9]. It should be noted that the literature has not yet reported the existence of ideal systems for controlled and targeted drug delivery.

Dezavantajul sistemelor cu eliberare controlata existente (medicamente) consta in faptul ca pentru a fi un transportor microstructurat sau submicrostructurat, chitosanul este eficient numai in forma protonata si solubila, facilitând astfel transportul paracelular al unor substanțe active hidrofile. Aceasta proprietate implica faptul ca chitosanul este eficient in absorbția si transportul substanțelor active antiinflamatoare intr-o porțiune limitata a tractului digestiv, in care valorile de pH sunt mai mici sau egale cu valoarea de 6,5 a preparatului [10, H]The disadvantage of existing controlled release systems (drugs) is that to be a microstructured or submicrostructured transporter, chitosan is effective only in protonated and soluble form, thus facilitating the paracellular transport of hydrophilic active substances. This property implies that chitosan is effective in the absorption and transport of anti-inflammatory active substances in a limited portion of the digestive tract, where the pH values are less than or equal to the value of 6.5 of the preparation [10, H]

Avantajul procedeului propus consta in autoasamblarea simultana prin utilizarea metodei de ultrasonare rezultând micro si submicrostructuri ale chitosanului cu substanțe active antiinflamatoare intr-o singura etapa. Materialul nano structurat obtinut este mai versatil, se poate utiliza ca film subțire, ca material spongios si ca microparticule obținute prin atomizare. Ca film subțire, produsul se folosește in aplicații topice avand rol de eliberare a medicamentului antiinflamator cat si de reconstrucție a pielii datorita chitosanului care are proprietatea de a fi atat biodegradabil, biocompatibil si citoprotector. Ca si material spongios se poate folosi fie ca atare fie presat si actioneaza ca hemostatic in procesele hemoragice dar si la eliberarea de medicament antiinflamator pentru răni deschise. Microparticulele pot fi formulate in tablete prin amestecare cu excipienti, produsul avand mare potențial in procesele antiinflamatorii ale tractului digestiv, când chitosanul actioneaza ca o bariera de protecție pe mucoasa tractului digestiv si este in același timp un protector fata de acțiunea agresiva a medicamentelor inflamatoare. Procedeul propus prin faptul ca permite realizarea intr-o singura etapa a ansamblelor micronice si submicronice de chitosan cu substanțe active antiinflamatoare scade semnificativ prețul de producție.The advantage of the proposed procedure is the simultaneous self-assembly by using the ultrasonic method resulting in micro and submicrostructures of chitosan with active anti-inflammatory substances in a single stage. The nano-structured material obtained is more versatile, it can be used as a thin film, as a spongy material and as microparticles obtained by atomization. As a thin film, the product is used in topical applications with the role of releasing the anti-inflammatory drug and rebuilding the skin due to chitosan which has the property of being both biodegradable, biocompatible and cytoprotective. As a spongy material it can be used either as such or pressed and acts as a hemostatic in hemorrhagic processes but also in the release of anti-inflammatory drug for open wounds. The microparticles can be formulated in tablets by mixing with excipients, the product having great potential in the anti-inflammatory processes of the digestive tract, when chitosan acts as a protective barrier on the mucosa of the digestive tract and is at the same time a protector against the aggressive action of inflammatory drugs. The process proposed by the fact that it allows the realization in a single stage of micron and submicron assemblies of chitosan with anti-inflammatory active substances significantly reduces the production price.

Nano si micro particulele de chitosan conform invenției prezintă si alte avantaje: tehnologie de realizare este simpla; consumul de reactivi, materiale si timp este mic; creste gradul de solubilitate si biodisponibilitate, imbunatateste stabilitatea moleculei bioactive, elimina gustul si/sau mirosul neplăcut, înlătură efectele agresive a medicamentelor antiinflamatoare asupra întregului tract digestiv.Nano and chitosan micro particles according to the invention have other advantages: the production technology is simple; the consumption of reagents, materials and time is low; increases the degree of solubility and bioavailability, improves the stability of the bioactive molecule, eliminates unpleasant taste and / or odor, removes the aggressive effects of anti-inflammatory drugs on the entire digestive tract.

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Procedeul propus constitue soluții noi aduse procesului de autoasamblare nano si micro particulelor de chitosan cu substanțe active antiinflamatorii. Materialele de tip compozit pe baza de polizaharide reprezintă o noua categorie de materiale biocompatibile si biodegradabile, care deschid multe si variate posibilități de explorare si exploatare a proprietăților lor. Prepararea nano- sau microparticolelor de chitosan, „incarcarea” acestora cu substanțe bioactive are loc prin complexare si/sau prin adsorbtie fizica.The proposed process is a new solution to the process of nano and micro self-assembly of chitosan particles with anti-inflammatory active substances. Polysaccharide-based composite materials represent a new category of biocompatible and biodegradable materials, which open many and varied possibilities for exploring and exploiting their properties. The preparation of nano- or microparticles of chitosan, their "loading" with bioactive substances takes place by complexation and / or by physical adsorption.

Se da in continuare un exemplu de realizare a invenției. S-au preparat soluții de acid acetilsalicilic (ASA), diclofenac - sarea sodica (DCF-Na) si hidrocortizon acetat (HcAc) prin dizolvare in condiții normale in apa cu alcool etilic in proporție 1:1, si s-au înglobat prin agitare in matricea de hidrogel. Concentrația finala a pricipiilor active antiinflamatoare in soluție este de 0,05% . Procesul de micro- sau nano-structurare este favorizat de adaugarea unor aditivi specifici, in cazul de fata au fost adaugati 2%v/v Tween-80 si 0,2% v/v acid oleic, sub continua agitare, timp de cca. 6 ore, la temperatura de 30°C. Micronizarea particulelor s-a făcut prin ultrasonare la 35 Hz in aparatul cu ultrasunete Elmasonic E 60 H. Intr-o prima etapa s-au realizat hidrogeluri conținând microparticule de chitosan cu acid acetil-salicilic (notat ASA), diclofenac - sarea sodica si (notat DCF - Na), si hidrocortizon acetat (notat HcAc), prin dizolvarea CTS in soluție slab acida. In final, hidrogelul “mama” (matrice) este omogen, de culoare galben pai. Materialul nano structurat se obține ca film subțire (prin depunere si uscare controlata la 30°C timp de 24h in vase Petri), ca material spongios (in aparatul de liofilizare CHRIST ALPHA 1-4 LD, la o temperatura de -58°C si presiune de 0,009 atm) sau ca microparticule obținute prin atomizare (in aparate de atomizare). Materialele solide au consistenta, aspect si proprietăți diferite, in funcție de compoziție.An embodiment of the invention is given below. Solutions of acetylsalicylic acid (ASA), diclofenac - sodium salt (DCF-Na) and hydrocortisone acetate (HcAc) were prepared by dissolving under normal conditions in water with ethyl alcohol in a ratio of 1: 1, and were incorporated by stirring. in the hydrogel matrix. The final concentration of active anti-inflammatory principles in the solution is 0.05%. The micro- or nano-structuring process is favored by the addition of specific additives, in this case 2% v / v Tween-80 and 0.2% v / v oleic acid were added, under continuous stirring, for approx. 6 hours, the temperature of 30 ° C. Micronization of the particles was done by ultrasound at 35 Hz in the Elmasonic E 60 H ultrasound apparatus. In a first stage, hydrogels were made containing microparticles of chitosan with acetyl-salicylic acid (noted ASA), diclofenac - sodium salt and - Na), and hydrocortisone acetate (denoted HcAc), by dissolving CTS in weakly acidic solution. Finally, the "mother" hydrogel (matrix) is homogeneous, straw yellow. The nano-structured material is obtained as a thin film (by controlled deposition and drying at 30 ° C for 24 hours in Petri dishes), as a spongy material (in the CHRIST ALPHA 1-4 LD lyophilizer, at a temperature of -58 ° C and pressure of 0.009 atm) or as microparticles obtained by atomization (in atomizing devices). Solid materials have different consistency, appearance and properties, depending on the composition.

Ansamblele micronice si submicronice de chitosan cu substanțe active antiinflamatoare astfel obținute au fost caracterizate morfologic prin microscopie optica (microscopul IR JASCO IRT-3000 - accesoriu al spectrometrului FTIR 6100) si prin FTIR. Ansamblele pe baza de chitosan si substanțe antiinflamatorii obyinute prin autoasamblare simultana sunt prezentate in Fig.l, iar materialele obținute cu aceste ansambluri sunt prezentate in Fig 2. întrucât latura fiecărei imagini înregistrate si redate in figura 1 este de 200 pm, se poate aprecia cu ușurința si se demonstrează ca vezicule bine delimitate de ansamble sunt formate, avand dimensiuni intre 0,5 pm si 30 pm.The micron and submicron assemblies of chitosan with anti-inflammatory active substances thus obtained were morphologically characterized by optical microscopy (IR JASCO IRT-3000 microscope - accessory of the FTIR 6100 spectrometer) and by FTIR. The assemblies based on chitosan and anti-inflammatory substances obtained by simultaneous self-assembly are shown in Fig. 1, and the materials obtained with these assemblies are shown in Fig. 2. Since the side of each image recorded and rendered in Figure 1 is 200 pm, it can be appreciated with ease and it is demonstrated that well-defined vesicles of assemblies are formed, having dimensions between 0.5 pm and 30 pm.

Procedeul de obținere a nanoansamblurilor pe baza de chitosan si substanțe antiinflamatorii contribuie esențial la implementarea de noi metode de prevenție și intervenționale neinvaziv, prin formularea de materiale noi (medicamente) cu eliberare controlata prin originalitatea materialului care se propune a fi elaborat, si prin aplicabilitatea imediata, cu efecte deosebite in imbunatatirea securității si calitatii vieții.The process of obtaining nanoassemblies based on chitosan and anti-inflammatory substances contributes significantly to the implementation of new methods of prevention and non-invasive intervention, by formulating new materials (drugs) with controlled release by the originality of the material to be developed, and by immediate applicability , with special effects in improving security and quality of life.

Claims

1. Process for obtaining functional assemblies of micron and submicron dimensions of chitosan characterized in that they have encapsulated active substances with anti-inflammatory properties by complexation and / or physical adsorption, which may lead to the formulation of new materials (drugs) with controlled release of the active substance.

Functional assemblies obtained by claim 1, characterized in that they are in the liquid aggregation state, contain chitosan aggregates with anti-inflammatory active substances which have been prepared by ultrasound at 35 Hz of chitosan hydrogels added with 2% v / v Tween-80 and 0.2% v / v oleic acid, at room temperature, in which solutions of anti-inflammatory active substances (acetyl-salicylic acid, diclofenac-sodium salt, hydrocortisone acetate) were previously added, which by simultaneous self-assembly allow the formation of solid materials of film type and spongy type.