Patent Application: US-88183107-A

Abstract:
the present invention relates to biocompatible and biodegradable stimuli - sensitive polymeric nanoparticles , which were formed by ion - ion interaction in aqueous media . synthetic and biological macromolecules with ionizable functional groups are capable of forming nanoparticles whose size and surface properties are sensitive to environmental influences such as ph , temperature and salt concentration . nanodevices are designed for therapeutic applications as drug and nucleic acid carriers , and / or for mri diagnosis as contrast agents . these nanodevices are designed for therapeutic applications as targeted drug carriers . additionally , they can be used as contrast agents for mri diagnosis . these nanosystems are also potential carriers for delivery of active ingredients as dna , rna , short interfering rna , antisense oligonucleotides , and triple helix forming oligonucleotides etc . for pharmaceutical applications . their adjustable size offers yet another advantage .

Description:
macromolecules with ionizable functional groups such as carboxyl , amino , etc ., in an aqueous medium form polycations and polyanions . under specific conditions polycations and polyanions form nanoparticles by ion - ion interactions . the formation of nanoparticles requires specific reaction parameters , otherwise flocculation and precipitation occurs . however , once the nanoparticles were formed at specific ph and salt concentration , the nanosystem is stable . ion - ion interaction can be performed between the functional groups of polyions , and the ratio of original polyions and the order of mixing can affect the particle structure and morphology . the linear polyelectrolyte chains can collapse in compact globules or can extend to coil conformations depending on the ph . the conformation of polymers is an important factor . the final formation of globular nanoparticles is dependent upon interactions between polyelectrolytes . core - shell or sandwich like morphology can be obtained by varying the ratio of original polyions , the ph and the order of mixing . fig1 shows a representation of nanoparticles formed by ion - ion interaction of polyelectrolyte macromolecules . a : positively charged polyelectrolyte ( dark blue lines ) on the surface . b : negatively charged polyelectrolyte ( light blue lines ) on the surface . the surface charge is determined by the sequence of mixing . fig2 is a schematic representation of nanodevice . the nanoparticle is loaded with dna or rna or sirna , as - on , tfo etc . and targeting molecules . the size of nanoparticles depends on the ph of the solution . the hydrodynamic diameter of nanoparticles increases by increasing the ph . surface charge of nanoparticles reveals the sequence of polyion addition . at lower ph , positively charged nanoparticles can be found , independently of the ratio of polyions or order of mixing . by increasing the ph , negatively charged nanoparticles are formed . the ratio of charged free functional groups determines the charge extent of nanoparticles , which depends on the ph and the ratio of functional groups . the hydrodynamic diameter and the stability of nanoparticles were investigated in kcl solution . it was found that the hydrodynamic diameters decreased with increasing salt concentration , but the stability of the aqueous solutions was independent of the salt concentration . the stability of the aqueous solution and the size of nanoparticles depend on the original concentration of polyions . the hydrodynamic diameter of nanoparticles increases with increasing the original concentration of polyions . the stability of the aqueous solution decreases with increasing the original concentrations , and precipitation can be observed in some cases of mixing at high concentration of original polyions . nanoparticles formed from poly acrylic acid ( paa ) and polyammonium salt ( pamm ) paa with mw = 200 kda and poly ( 2 - methacryloxyethyltrimethylammonium bromide ) were dissolved in water at a concentration of 1 mg / ml . the ph value of solutions was adjusted to ph 3 by 0 . 10 mol / dm 3 sodium hydroxide . the solution of pamm was added to the solution of paa with gentle stirring . after 1 hour the ph was increased to 7 resulting in a stable nanosystem with particle size of 50 to 350 nm measured by laser light scattering method . the size of nanoparticles is variable in a range of 10 - 1 , 000 nm by using polymers with different molecular weight . also the particle size increased at higher ph due to the repulsion of negative charges . nanoparticles formed from chitosan ( chit ) and poly gamma glutamic acid ( pga ) chit with mv = 320 kda and pga with mw = 1 . 3 mda were dissolved in distilled water . the concentration was varied in the range 0 . 1 mg / ml - 2 . 0 mg / ml . the ph value of the solutions was adjusted to ph = 3 with 0 . 10 mol / dm 3 hydrochloric acid . the ratio of polyelectrolyte and the order of mixing were modulated . after 1 hour mixing , the ph was increased with 0 . 1 m sodium hydroxide solution resulting in stable nanosystems . the hydrodynamic diameter of nanoparticles was in the range of 40 - 480 nm at ph 3 , and at ph 7 was 470 - 1300 nm measured by laser light scattering method . there was some precipitation at higher ph caused by flocculation and coagulation . the size of nanoparticles can be varied by using polymers with different molecular weights . chit with mv = 320 kda and hyal with mw = 2 . 5 mda were dissolved in water . the concentration of chit was varied in the range 0 . 1 mg / ml - 1 . 0 mg / ml , and of hyal 0 . 04 - 0 . 2 mg / ml . the ph value of solutions was adjusted to ph 3 with 0 . 10 mol / dm 3 hydrochloric acid . the ratio of polyelectrolyte and the order of mixing were modulated . after 1 hour mixing the ph was increased with 0 . 1 m sodium hydroxide solution resulting in stable nanosystems . the hydrodynamic diameter of nanoparticles was in the range of 130 - 350 nm at ph 3 , and was higher than 600 nm at ph 7 as measured by laser light scattering . there was some precipitation at higher ph caused by flocculation and coagulation . the size of nanoparticles can be varied by using polymers with different molecular weights . chit with mv = 320 kda and alga with mv = 30 kda were dissolved in water . the concentration of chit was varied in the range 0 . 1 mg / ml - 1 mg / ml , and of alga 0 . 04 - 0 . 2 mg / ml . the ph value of solutions was adjusted to ph = 3 by 0 . 10 mol / dm 3 hydrochloric acid . the ratio of polyelectrolyte and the order of mixing were modulated . after 1 hour mixing the ph was increased by 0 . 1 m sodium hydroxide solution resulting stable nanosystems at a ph = 3 . there was some precipitation at higher ph caused by flocculation and coagulation . the size of nanoparticles can be varied by using polymers with different molecular weights . chitosan was partially modified by betaine . the modification was performed by using the carbodiimide technique . chit was dissolved in hydrochloric acid media . betaine was dissolved in water and then adjusted to ph 6 . 5 with 0 . 1 m sodium hydroxide solution . water soluble carbodiimide was added to the betaine solution and the reaction was stirred for 30 min and subsequently mixed with the chitosan solution . the modified chit and pga with a mw = 1 . 3 mda were dissolved in water . the concentration was varied in the range 0 . 1 mg / ml - 2 . 0 mg / ml . the ph values of the solutions was adjusted to ph = 3 with 0 . 10 mol / dm 3 hydrochloric acid . the ratio of polyelectrolyte and the order of mixing were modulated . after 1 hour mixing the ph was increased by 0 . 1 m sodium hydroxide solution resulting in stable nanosystems . there was some precipitation at higher ph caused by flocculation and coagulation . the size of nanoparticles can be varied by using polymers with different molecular weight . to assess the suitability of chit / pga nanosystem for intracellular delivery of bioactive compounds including nucleic acids , first additional components were incorporated to allow e . g . cancer cell specific targeting and detection of cellular uptake . as a targeting moiety , the vitamin folic acid ( fa ) was chosen , which has a high affinity for folate receptors ( far ) which are overexpressed in a number of epithelial and myeloid cancer cells . fa was conjugated to poly - γ - glutamic acid ( mw 1 . 3 mda , gpc ) using water soluble carbodiimide . after the dropwise addition of edc ( 1 - ethyl - 3 -[ 3 - dimethylaminopropyl ] carbodiimide hydrochloride , 8 mg in 1 ml dd . water ) to the γ - pga solution ( 50 ml , 1 mg / ml , ph 6 . 5 ), the reaction mixture was stirred at room temperature for 30 min . fa ( 12 mg in dmso ) was added and stirred at room temperature for 24 h . the γ - pga - fa conjugate was purified by dialysis and the number of fa molecules per γ - pga was estimated by uv - vis absorption spectroscopy ( λ max1 368 nm , ε9120 ; λ max2 283 nm , ε25100 ). this showed that an average of 7 fa molecules was attached to one pga molecules by this method . low molecular weight chitosan ( mw 320 , 000 da , as determined by viscosity measurements , and with a degree of deacetylation of 88 %) solution ( 10 ml , 1 mg / ml in water , solubilised with hcl and ph adjusted to 6 . 5 with naoh ) was mixed with an aliquot of fluorescein isothiocyanate ( fitc , 1 mg / ml in dmso , 250 μl ) and the reaction mixture was stirred at room temperature for 24 h . fluorescein - labelled chitosan ( chit - fitc ) was purified by dialysis against water ( mw cutoff 10 000 da , 3 days ) and characterised by uv - vis spectrophotometry , which showed that 71 fluorescein molecules were attached per chitosan molecules by this method . stable self - assembled polyelectrolytes were developed via an ionotropic gelation process between the folated γ - pga and the fluorescently labelled chitosan linear chains . when an equal volume of aqueous γ - pga - fa ( 0 . 3 mg / ml , ph 9 . 0 ) and chit - fitc ( 0 . 3 mg / ml , ph 4 . 0 ) were mixed under continuous stirring , an opaque colloidal system was formed ( 75 % transmittance at λ500 nm , ph 7 . 4 ), which remained stable at room temperature for several weeks at physiological ph . the presence of individual nanoparticles was confirmed and their size distribution characterized by transmission electron microscopy ( tem ), atomic force microscopy ( afm ) and dynamic light scattering ( dls ) as described before . the analyses demonstrated that the chit - fitc / γ - pga - fa nanosystem consists of spherical particles with a smooth surface both in aqueous environment and in a dried state ( fig3 a , b ). tem micrograph showed a size range of 30 - 160 nm with a mean value of 67 . 8 nm ( fig3 b ), while dls reported a bimodal distribution for hydrodynamic diameter ranging between 70 - 90 nm and 160 - 200 nm with mean values of 80 and 178 nm , respectively ( fig3 c ). this is consistent with the particles swelling in an aqueous environment . the overall charge ratio of the nanoparticles ( number of — nh 3 + groups of chit vs — coo − groups on γ - pga ) was calculated as + 0 . 67 :− 1 based on the weight ratio between chit and γ - pga . this results in a negative zeta - potential at physiological ph , which may contribute to stabilisation of the nanosystem via charge repulsion between individual particles ( majoros et al ., 2006 ; hong et al ., 2007 ; hajdu et al ., 2007 ; berger et al ., 2004 ; hsieh et al ., 2005 ; lin et al ., 2006 ; lin et al ., 2007 ). fig3 shows the characterisation of chitosan - fitc / poly - γ - glutamic acid - folate nanoparticles ( chit : γ - pga 1 : 1 , 0 . 3 mg / ml ). ( a ) afm micrograph with color key for the third dimension , ( b ) tem micrograph of particles and particle size distribution , and ( c ) the size distribution of the particles was determined by dls . chit with mv = 320 kda was dissolved in water at ph = 3 . an aqueous solution of dna with mw = 32 kda and with specific sequence was added . a stable nanosystem was formed . in the second step , pga with mw = 1 . 2 mda was added to cover the residual surface . the sandwich - like composite nanodevice containing the dna molecules was stable at ph = 7 and the nacl concentration was 0 . 1 g / dm 3 . nanoparticles were formed from chit , dna and pga by a general method represented in fig4 . fig4 shows the preparation of nanodevice for dna / rna / sirna , antisense oligonucleoties etc delivery . polycation ( pc ) is complexed with dna then coated with a polyanion ( pa ), via an ionotropic gelation process . pc is labelled with fluorescein isothiocyanate ( fitc ) and single stranded dna was labeled with cy3 fluorescent dye for microscopic imaging . pa was conjugated with folic acid as cancer cell specific targeting moiety . chit with mv = 320 kda was labelled with fitc as described in example 6 . pga with mw = 1 . 2 mda was conjugated with folic acid ( fa ) as described in example 6 . single stranded dna consisting of 20 nucleotides and with a specific sequence was labelled at the 3 ′ end with cy3 fluorescent dye ( dna - cy3 ). chit - fitc was dissolved in water at a concentration of 0 . 3 mg / ml and at ph 4 . dna - cy3 was dissolved in water at a concentration of 0 . 6 mg / ml and at ph 7 . 4 . pga - fa was dissolved in water at a concentration of 0 . 3 mg / ml and at ph 9 . 5 . nanoparticles were formed by either mixing 1 ml of chit - fitc solution pre - combined with 50 μl dna - cy3 solution and 1 ml of pga - fa solution or by mixing 1 ml of chit - fitc solution and 1 ml of pga - fa solution pre - combined with 50 μl dna - cy3 solution . the nanosystems were characterised by dynamic light scattering ( dls ) or atomic force microscopy ( afm ). the results showed that both types of nanoparticles ( which differ in the order in which components were mixed ) had an effective diameter of 75 - 77 nm in aqueous environment ( by dls with a distribution between 46 - 148 nm ) or up to 31 nm in a dried state ( by afm ) as shown in fig5 . fig5 shows an atomic force microscopic image of chit - fitc / dna - cy3 / pga - fa nanoparticles . nanoparticles from example 8 were tested for intracellular delivery of single stranded oligonucleotides into the human cervical cancer cell line , hela , by laser scanning confocal microscopy ( lscm ). cells were cultured in rpmi 1640 medium ( sigma , st . louis , mo .) supplemented with 10 % fetal bovine serum ( fisher chemicals , fairlawn , n . j .). cells were grown at 37 ° c . in a humidified atmosphere of 5 % co 2 ( v / v ) in air . all experiments were performed on cells in the exponential growth phase . a culture of the cells were incubated with the nanoparticles for up to 30 min and the internalisation of the oligonucleotide ( dna - cy3 ) as well as the chit - fitc was observed . the experiments clearly showed that the dna was delivered into the cytoplasm of hela cells ( fig6 ). the form of entrance appeared to be vesicular giving rise to an uneven distribution of the dna within the cytoplasm . after longer incubation , the dna - cy3 compound showed accumulation within the nucleus of the cells ( fig6 min ). in control experiments , performed by exposing the cells to dna - cy3 without encapsulation within the nanoparticles showed that the dna did not accumulate within the cells or nucleus . fig6 shows nanoparticle assisted accumulation of the labelled oligonucleotide dns - cy3 in hela cells during 1 , 3 , 6 and 10 minutes of incubation . combined signals of visible , fluorescein ( green ) and cy3 ( red ) from laser scanning confocal microscopy . accordingly , a chitosan / poly - γ - glutamic acid - based self - assembling nanoparticulate system as a delivery platform for nucleic acids is provided . the two main components of this polycationic - polyanionic gel - type composite nanosystem are renewable as chitosan is derived from chitin of crustacean shell by alkaline deacetylation while γ - pga is also easily obtained from bacillus sp . ferments , where it is produced as slime . cs and γ - pga are known to be fully biocompatible , biodegradable and likely non - immunogenic and also failed to display any toxicity in our cellular and in vivo studies . also , the two polymers have a wide range of biomedical applications in separate or in combination . additional advantages of the cs / γ - pga nanosystem as nanocarrier are that ( i ) its self - assembling nature provides simple preparation without resorting to chemical cross - linking , organic solvents , or other toxic additives ( ii ) the use of degraded polymers facilitate particle size control , and that ( iii ) surface charge and functionality of nps are conveniently tunable by varying component mixing ratios . thus , it will be seen that the advantages set forth above , and those made apparent from the foregoing description , are efficiently attained and since certain changes may be made in the above construction without departing from the scope of the invention , it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense . it is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described , and all statements of the scope of the invention which , as a matter of language , might be said to fall therebetween . now that the invention has been described , barron - 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