Patent Application: US-201515512288-A

Abstract:
the present invention provides a nano - particle based structure or composition for a combinational cancer therapy . the structure has a doxorubicin physically loaded on core - shell silver polymeric nanoparticles with a ratio of 3 . 3 - 5 . 5 % doxorubicin to 1 % silver to 2 - 10 % polymer . this structure enhances the cellular uptake of dox in comparison to the current conventional combination therapy . the dox - loaded nano - particles result in an improved the therapeutic efficiency of dox , and reduced its toxicity , which cannot occur in case of adding dox and agnps .

Description:
the present invention entails the development of ( nps )- based combinatorial therapy composed of dox - loaded on core - shell silver / polymeric ( pva , peg , and pvp ) nps . this nps - based combinatorial therapy is based on combining core - shell silver / polymeric ( pva , peg , and pvp ) nanoparticles , that it has an anticancer effect along with dox aimed at achieving maximum therapeutic efficacy , while minimizing dox &# 39 ; s dose and systemic toxicity . the aim of this invention is to formulate a nps - based combinatorial therapy that could ( 1 ) provide combination therapy possessing synergic anticancer action , ( 2 ) provide passive cancer targeting mechanism ( which can selectively target and kill cancer cells without harming the neighboring normal cells ), ( 3 ) improve pharmacokinetics profile of dox , ( 4 ) improve therapeutic efficacy , ( 5 ) reduce dox &# 39 ; s dose , and ( 6 ) dox &# 39 ; s toxicity . the nps - based combinatorial therapy according to an embodiment of the present invention ( fig1 ) can be been prepared as follows : ( 1 ) preparation of silver nanoparticles coated with fda - approved synthetic polymers ( pva , peg and pvp ), ( 3 ) in - vitro testing of free dox alone , an individual type of ag / polymeric ( pva , peg and pvp ) nps alone , and dox - ag / polymeric ( pva , peg and pvp ) nanocarriers ( ncs ) on breast cancer cell line ( mcf - 7 ) and the cell viability and 50 % inhibition concentration ( ic50 ) were measured after 48 hrs incubation . the in - vitro test was conducted using the mtt assay on breast cancer cell line ( mcf - 7 ) and human fibroblast cell line ( 1br htert ), which was performed as follows : ( 1 ) testing the cytotoxic effect of different concentrations ( 2 , 4 , 8 , 10 , and 12 μg / ml ) of free dox alone , ( 2 ) testing the cytotoxic effect of agnps and core - shell ag / polymeric ( pva , peg , and pvp ) nps using different concentrations ( 10 , 20 , 50 , and 100 μg / ml ), and ( 3 ) testing the cytotoxic effect of nps - based combinatorial therapy of an individual type of dox - ag / polymeric ncs including dox - loaded ag / pva ncs , dox - loaded ag / peg ncs , and dox - loaded ag / pvp ncs using different concentrations ( 0 . 1 , 0 . 2 , and 1 μg / ml dox ). a prior art example to contrast the embodiments of the present invention hekmat et al . in - vitro examined the combination effect of commercially available ethylenglycole - stabilized agnps ( purchased from bio - cera co , ltd , south korea ) and dox on breast cancer cell line ( mcf - 7 ). the in - vitro mtt test was performed as follows : ( 1 ) testing the cytotoxic effect of different concentrations of agnps alone ( 1 . 7 , 2 . 55 , 5 , 8 . 5 , 11 . 9 , and 20 . 4 μg / ml ), ( 2 ) testing the cytotoxic effect of different concentrations of free dox ( 0 . 0725 , 0 . 145 , 0 . 232 , 0 . 319 , 0 . 58 , and 0 . 725 μg / ml ), and ( 3 ) testing the cytotoxic effect of agnps in combination with dox of the following concentrations ( 0 . 174 and 0 . 232 μg / ml for dox ) and ( 1 . 7 and 2 . 55 μg / ml for agnps ) ( this combination was done by conventional addition ), and then the cell viability and ic 50 were measured following 48 hrs incubation [ 20 ]. after 48 hrs incubation , the cell viability was measured and the results were plotted in graphs ( fig2 a - d ). from these graphs , ic 50 were determined to be 48 μg / ml for agnps and & gt ; 100 μg / ml for ag / pva nps and ag / peg nps , and 42 μg / ml for ag / pvp nps ( table 1 ). basically , agnps possess an anticancer action owing to their potential to translocate at the mitochondria and nucleus where releasing ag + ions . the released ag + ions trigger the generation of ros mediating oxidation stress . the oxidation stress causes a series of cellular events including reduced the levels of glutathione ( gsh ) and superoxide dismutase ( sod ), and elevated lipid peroxidation , which eventually lead to dna damage and cancer cell death [ 21 ]. the difference in in - vitro results among different uncoated and coated agnps is mainly ascribed to the effect of polymeric coating . it has been well documented that nps &# 39 ; surface coating controls agnps &# 39 ; dissolution , which is directly correlated with their cytotoxicity [ 22 - 24 ]. results revealed that ag / pvp nps exhibited the highest cytotoxicity ( ic 50 : 42 μg / ml ) as compared to agnps ( ic 50 : 48 μg / ml ), core - shell ag / polymeric ( pva and peg ) nps ( ic 50 : above 100 μg / ml ). dobias and bernier - latmani reported that core - shell ag / pvp nps exhibited higher cytotoxic effect than agnps because ag / pvp nps exhibit an order of magnitude higher dissolution as compared to agnps . ag / pvp nps exhibit a higher dissolution rate because pvp polymer is non - charged , therefore , the detached pvp chains could not reduce ag + ions and thus resulted into higher cytotoxic effect . however , the slow dissolution of agnps was ascribed to the ability of the carboxylic group of citrate to bind to ag + ions , hence reducing ag + to ag 0 and decreasing agnps cytotoxicity [ 21 ]. however , ag / pva , and ag / peg nps showed a minimal cytotoxic effect when compared to agnps and ag / pvp nps , owing to their higher stability and slower dissolution rate . luo et al . also revealed that core - shell ag / peg nps and ag / pva nps exhibit a very slow dissolution owing to the binding of detached negatively charged peg and pva polymer chains with released ag − ions , forming stable ag - ligand complexes resulting in ag + ions retention and decrease in cytotoxicity [ 26 ]. by comparing these results with the in - vitro results of agnps obtained from hekmat et al . study , it was found that ic 50 of agnps was 55 μm equal to 9 . 35 μg / ml ( table 1 ). these results revealed that the used agnps are unstable and exhibit faster dissolution rate as compared to the invented agnps ; since 100 μg / ml of the invented ag / polymeric ( ag / pva and ag / peg ) nps did not reach the same cytotoxic effect reached by 9 . 35 μg / ml of the ready - made agnps . the cell viability was measured after 48 hrs incubation with free dox alone and nps - based combination therapy . the results obtained from mtt assay were plotted in graphs ( fig3 a - e ), the ic 50 was found to be 3 . 7 μg / ml for free dox alone , 1 - 11 . 23 μg / ml for dox - agncs , 0 . 19 - 3 . 4 μg / ml for dox - ag / pva ncs , 0 . 14 - 3 μg / ml for dox - ag / peg ncs , and 0 . 1 - 3 . 5 μg / ml for dox - ag / pvp ncs . these results revealed that the ic 50 of dox - ag / polymeric ( pva , peg and pvp ) ncs was achieved at a much lower dose of dox and ag , as compared to dox - agncs , thus indicating the superiority of dox - ag / polymeric ( pva , peg and pvp ) ncs . the results also revealed that dox - ag / polymeric ( pva , peg and pvp ) ncs showed ic 50 at 10 - fold reduced doses compared to free dox alone . in addition to the low concentration of dox , the ic 50 was achieved at very low concentration of nps of 3 . 5 μg / ml for ag / pvp nps , 3 . 4 μg / ml for ag / pva nps , and 3 μg / ml for ag / peg nps , respectively . taken together , the in - vitro results revealed that dox - ag / polymeric ( pva , peg and pvp ) ncs achieved the same efficacy of dox alone , but with 95 % reduced dose of dox . the achieved synergic anticancer effect of dox - ncs could be ascribed to two reasons : ( i ) the combined effect occurred due to the combination of both the cytotoxic effect of agnps along with the therapeutic effect of dox , and ( ii ) the enrichment in internalization of dox - ag / polymeric ncs via endocytosis allowing the release of dox inside the cell as compared to the passive diffusion mechanism of free dox into the cells . venkatpurwar et al . reported a significant enhancement in cytotoxicity of dox - auncs on human glioma cell line ( ln - 229 ) compared to free dox , possibly through enhanced cellular internalization owing to aunps mediated endocytosis [ 27 ]. chen et al . also reported the passive intracellular accumulation of methotrexate - auncs confirming auncs mediated endocytosis followed by methotrexate release inside cancer cells [ 28 ]. on the other hand , the in - vitro results obtained by hekmat et al . showed that the ic 50 of conventional combination therapy between agnps and dox was 15 μm agnps + 0 . 4 μm dox , which is equal to 0 . 23 μg / ml dox + 2 . 55 μg / ml agnps . by comparing ic 50 results of the invented nps - based combination therapy and conventional combination therapy present in the prior art ( hekmat et al . ), it was clearly observsed that results of nps - based combinatorial therapy showed a synergic anticancer effect similar to the prior art , but at a much lower dose of dox . as shown in table 2 , nps - based combinatorial therapy achieved an ic 50 but with around 40 % reduced dose of dox compared to dox - ag combination present in the prior art . the main difference between the invented nps - based combination therapy and the combination therapy present in the prior art , is the type of combination . in embodiment of this invention , the combination was based on loading nps with a chemotherapeutic agent , dox , while the combination in the prior art is based on simply adding agnps followed by dox ( without loading ). however , in this invention , dox was is physcially loaded on agnps through van der waal bond . the large surface area of agnps offers a large loading capacity for dox , which in turn results in enhancing the cellular uptake of dox resulting in the imporved therapeutic efficiency in comparison to the prior art . one can conclude that nps - based combinatorial therapy possesses a synergism at a much lower dose of dox , owing to the advantages of nanotherapeutics that include : passive cancer targeting and enrichment of cellular internationalization of drug via endocytosis — in comparison to the passive diffusion of free drug and combination therapy in the prior art . in addition , the dox - core - shell ag / polymeric ncs showed low toxicity on normal human fibroblast ( 1br htert ) cells . therefore , the dox dose can be reduced using this platform and in turn reduces its dose - dependent toxicity and adverse side effects . the size and morphology of the prepared agnps and core - shell ag / polyeric nps were characterized by sem , image j analysis software and ltv - visible spectroscopy . sem images showed that the prepared agnps and core - shell ag / polymeric nps were spherical , mono - dispersed and well - dispersed ( fig4 a - g ). the size distribution histogram obtained from the image j analysis software , showed that the average sizes of agnps and core - shell ag / polymeric ( pva , peg , and . pvp ) nps were 7 . 3 ± 1 . 8 nm , 6 . 1 ± 2 . 8 mn , 8 . 4 ± 5 . 4 tun and 13 . 3 ± 7 . 1 nm ( fig4 b , 4d , 4f , and 4h ), respectively . the uv - vis spectra of agnps , ag / pva nps and ag / peg nps ( fig4 a - d ) showed a sharp surface plasmon resonance ( spr ) peak at ˜ 400 nm , which is the characteristic peak of spherical , mono - dispersed and well - dispersed agnps [ 29 - 30 ]. however , the uv - vis spectrum of ag / pvp nps ( fig4 d ) showed a sharp peak at 420 nm . previous studies demonstrated that spherical and mono - dispersed ag / pvp nps of size ranging between 10 - 20 nm display a spr band at ˜ 412 - 437 nm [ 31 ]. the ft - ir spectra also confirmed the formation of agnps and core - shell ag / polymeric ( pva , peg , and pvp ) nps [ 31 - 34 ]. see also fig1 - 13 . following synthesis of ag / nps and core - shell ag / polymeric nps , each individual type of np was loaded with dox . the drug loading efficiency was determined based on dox content in the supernatant . the drug loading efficiency percentages were determined to be : 58 . 3 %, 54 . 9 %, 56 . 5 % and 62 . 5 % for : agnps , core - shell ag / pva ncs , core - shell ag / peg ncs and ag / pvp ncs , respectively . the bond between dox and nps was detected using uv - vis spectra ( fig5 a - d ) and sem ( fig6 a - h ). the uv - vis spectra ( fig5 a - d ) indicated that binding of dox to nps resulted in a red shift of the spr peak of loaded nps from 400 to ˜ 500 nm . since the release behavior of dox - ncs at the desired site is of a great importance for formulating an ideal cancer - targeted drug delivery system , in - vitro release studies were performed at two different ph values were tested : ph 7 . 4 , which mimics the ph of the blood stream and ph 5 , which mimics the ph of the endosomes within cancer cells . in - vitro results ( fig6 a - b ) that dox - agncs , dox - ag / pva ncs , dox - ag / peg ncs and dox - ag / pvp ncs released 96 . 6 %, 97 . 4 %, 98 % and 96 . 4 % of dox at ph 5 . while at ph 7 . 4 , the release percentages of dox were 73 . 4 %, 54 . 3 %, 59 . 8 % and 68 . 5 % over the course of 6 hrs . on the other hand , free dox solution was also used as a control and it was found that free dox released 97 . 4 % of dox at ph 5 , and 67 . 7 % at ph 7 . 4 over 4 hrs . in - vitro release is shown of free dox , and dox - ncs in tris - hcl buffer ph 5 ( fig7 a ) and pbs ph 7 . 4 ( fig7 b ). effect of agnps and core - shell ag / polymeric on mcf - 7 cells and 1br htert cells to assess the cytotoxic effect of agnps and core - shell ag / polymeric nps , mcf - 7 and 1br htert cells were exposed separately to different concentrations of nps for 48 hrs . agnps and core - shell ag / polymeric ( pva , peg and pvp ) nps decreased the cell viability of mcf - 7 cells and 1br htert cells ( fig8 a - d ) in a dose - dependent manner . the inhibitory concentration ( ic 50 ) was estimated to be 48 μg / ml for agnps , 42 μg / ml for ag / pvp nps and greater than 100 μg / ml for both ag / pva nps and ag / peg nps on mcf - 7 cells . the ic 50 of nps in 1br htert cells was 100 μg / ml for ag nps , ag / pva nps , and ag / peg nps , and 50 μg / ml for ag / pvp nps . the ag / pva and ag / pvp nps were more cytotoxic on cancer cells at the high concentration of 100 μg / ml , with ag / pvp nps being more cytotoxic on mcf - 7 cancer cells ( fig8 a - d ). effect of dox - core - shell ag / polymeric nps on mcf - 7 cells and 1br htert cells to investigate the cytotoxic effect of nps - based combinatorial therapy , first , different concentrations of free dox ( 2 , 4 , 8 , 10 , and 12 μg / ml ) were tested on mcf - 7 and 1br htert cells , and cell viability was determined after 48 hrs . ic 50 of free dox on mcf - 7 cells was determined to be 3 . 7 μg / ml ( fig1 ). based on the ic 50 of free dox , lower dox - ncs concentrations than the calculated ic 50 of free dox were selected ( 0 . 1 , 0 . 2 , and 1 μg / ml dox ) in order to assess whether the combination between dox and nps will induce synergism or not . the estimated ic so values of dox - agncs , dox - ag / pva ncs , dox - ag / peg ncs and dox - ag / pvp ncs on mcf - 7 cells were 1 . 00 - 11 . 23 μg / ml , 0 . 19 - 3 . 40 μg / ml , 0 . 14 - 3 . 00 μg / ml , and 0 . 10 - 3 . 50 μg / ml , respectively ( fig9 a - d ). on the other hand , the estimated ic so values of dox - ncs on 1br htert cells were 1 . 00 - 11 . 23 μg / ml for dox - agncs , ncs , dox - ag / peg ncs , and dox - ag / pvp ncs , while it was estimated to be 0 . 60 - 9 . 00 μg / ml for dox - ag / pva ncs ( fig9 a - d ). all dox - loaded core - shell ag / polymeric ncs were more cytotoxic on cancer cells than normal cells . notably , dox - ag / pvp combination was more cytotoxic than all three and was more cytotoxic on cancer cells . in conclusion , mono - dispersed spherical agnps and core - shell ag / polymeric ( pva , peg , and pvp ) nps were successfully synthesized , loaded with dox and in - vitro drug release of each individual type of dox - ncs was investigated . moreover , individual unloaded - nps , free dox and dox - ncs were tested for in - vitro cytotoxicity on mcf - 7 cells and 1br htert cells . in - vitro mtt experiments demonstrated that core - shell dox - ag / polymeric ncs at much lower doses showed a synergic cytotoxic effect towards mcf - 7 cells , and a lower cytotoxic effect on normal 1br htert cells . 2 . ferlay , j ., i . soerjomataram , and m . ervik , globocan 2012 v1 . 0 , cancer incidence and mortality worldwide : iarc cancer base no . 10 [ internet ], international agency for research on cancer , 2013 . 2012 . 3 . siegel , r ., d . naishadham , and a . jemal , cancer statistics , 2013 . ca : a cancer journal for clinicians , 2013 . 63 ( 1 ): p . 11 - 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