Nanoparticle-based Combinatorial Therapy

Abstract

The present invention provides a nano-particle based structure or composition for a combinational cancer therapy. The structure has a doxorubicin (DOX) physically loaded on core-shell silver polymeric nanoparticles (AgN-Ps) 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.

Claims

1. A nano-particle based composition for a combinational therapy, comprising: 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.

2. The composition as set forth in claim 1, wherein the core-shell silver polymeric nanoparticles comprises Ag as a core and Poly-Vinyl Alcohol (PVA), Poly-Ethylene Glycol (PEG), or Poly-Vinyl Pyrrolidone (PVP) as a shell.

3. The composition as set forth in claim 1, wherein each nanoparticle has a width of 5 nm to 20 nm.

4. The composition as set forth in claim 1, wherein the core-shell silver-polymeric nanoparticle has a width of 20 nm to 40 nm.

5. The composition as set forth in claim 1, wherein the concentration of the doxorubicin in the composition is less or equal to 0.2 μg/ml.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows according to an exemplary embodiment of the invention a schematic illustration of the NPs-based combination therapy. It further shows a study workflow and a mechanism of action behind the resultant synergic cytotoxic effect of DOX-loaded Ag/polymeric NCs at very low doses of DOX on MCF-7 cells. (Aspect 1) Preparation of NPs, (Aspect 2) DOX loading and (Aspect 3) Ag.sup.+ ions and DOX intracellular release possibly leads to mitochondrial dysfunction that generates ROS leading to DNA fragmentation and cell death.

[0024] FIGS. 2A-D show according to an exemplary embodiment of the invention cell viability percentage of MCF-7 cells following 48 hrs incubation with different concentrations of Ag NPs (FIG. 2A), Ag/PVA NPs (FIG. 2B), Ag/PEG NPs (FIG. 2C) and Ag/PVP NPs (FIG. 2D), respectively. All in-vitro results showed that cell viability decreased in a dose-dependent manner.

[0025] FIGS. 3A-E show according to an exemplary embodiment of the invention cell viability percentage of MCF-7 cells after 48 hrs incubation with different concentrations of free DOX (FIG. 3A), DOX-AgNCs (FIG. 3B), DOX-Ag/PVA NCs (FIG. 3C), DOX-Ag/PEG NCs (FIG. 3D), and Ag/PVP NCs (FIG. 3E), respectively. All in-vitro results showed that cell viability decreased in a dose-dependent manner.

[0026] FIGS. 4A-H show according to an exemplary embodiment of the invention in FIGS. 4A, C, E and G representative SEM images of AgNPs, and core-shell Ag/polymeric NPs and in FIGS. 4B, D, F and H size and frequency histograms of AgNPs and core-shell Ag/polymeric NPs.

[0027] FIGS. 5A-D show according to an exemplary embodiment of the invention UV-Vis spectra of: DOX-AgNCs (FIG. 5A) and DOX-core-shell Ag/Polymeric (PVA, PEG, and PVP) NCs (FIG. 5B, FIG. 5C, and FIG. 5D). Each UV-Vis spectrum showed a comparison between UV-Vis spectra of NPs, DOX and DOX-NCs

[0028] FIGS. 6A-H show according to an exemplary embodiment of the invention SEM images of NPs without DOX (FIGS. 6A, C, E and G) and DOX-Ag NCs and DOX-Ag/Polymeric NCs (FIGS. 6B, D, F and H), respectively.

[0029] FIGS. 7A-B show according to an exemplary embodiment of the invention in-vitro release of free DOX, and DOX-NCs in Tris-HCl buffer pH 5 (FIG. 7A) and PBS pH 7.4 (FIG. 7B).

[0030] FIGS. 8A-D show according to an exemplary embodiment of the invention the percentage of viable MCF-7 cells (810) and 1BR hTERT cells (820) as determined by the MTT assay following 48 hrs incubation with concentrations of 0, 10, 20, 50 and 100 μg/ml of: (FIG. 8A) AgNPs, (FIG. 8B) Ag/PVA NPs, (FIG. 8C) Ag/PEG NPs and (FIG. 8D) Ag/PVP NPs. The data are presented as a mean of at least three independent experiments (mean±SD). P values were calculated for each concentration between the two cell lines, and denoted if found to be significant (*P<0.05, **P<0.01 and ***P<0.001).

[0031] FIGS. 9A-D show according to an exemplary embodiment of the invention the percentage of viable MCF-7 cells (910) and 1BR hTERT cells (920) as determined by the MTT assay following 48 hrs incubation with concentrations of 0, 0.1, 0.2 and 1 μg/ml (concentrations are referring to DOX concentration) of: (FIG. 9A) DOX-Ag NCs, (FIG. 9B) DOX-Ag/PVA NCs (FIG. 9C) DOX-Ag/PEG NCs, and (FIG. 9D) DOX-Ag/PVP NCs. The data are presented as a mean of at least three independent experiments (mean±SD). P values were calculated for each concentration between the two cell lines, and denoted if found to be significant (*P<0.05, **P<0.01 and ***P<0.001).

[0032] FIG. 10 shows according to an exemplary embodiment of the invention FTIR spectra of silver nitrate, sodium citrate, and AgNPs. The FT-IR spectra of AgNPs, trisodium citrate and AgNO.sub.3 confirm the formation of AgNPs. By comparing the FT-IR spectra of AgNPs, sodium citrate and AgNO.sub.3, the FT-IR spectra of AgNPs showed a band at 3423.6 cm.sup.−1 that is not present in the FT-IR spectrum of AgNO.sub.3. This band is attributed to O—H stretching between AgNPs and trisodium citrate. In addition, it was found that the band at 1592.8 cm.sup.−1 is assigned for symmetric carboxylic group stretching mode of sodium citrate underwent a blue shift and appeared sharply at 1401 cm.sup.−1 in the spectrum of AgNPs and thus confirming the stabilization of AgNPs by carboxylic group of trisodium citrate. Moreover, the spectrum of AgNO.sub.3 displays a band at 1380 cm.sup.−1 corresponding to ion pair Ag.sup.+NO.sub.3.sup.− that is not found in the spectrum of AgNPs due to the separation of NO.sub.3 from its Ag.sup.1 counterpart.

[0033] FIG. 11 shows according to an exemplary embodiment of the invention FTIR spectra of Pure PVA and core-shell Ag/PVA NPs. The FT-IR spectra of pure PVA and core-shell Ag/PVA NPs are found to be nearly similar. Both spectra show a broad band at 3400 cm.sup.−1 for pure PVA and 3350 cm.sup.−1 for core-shell Ag/PVA NPs respectively, which corresponds to the stretching vibration of hydroxyl group of PVA. Both FT-IR spectra show a band at 2942 cm.sup.−1 is assigned to CH.sub.2 asymmetric stretching vibration. The bands at 1711cm.sup.−1 for pure PVA and 1709 cm.sup.−1 for core-shell Ag/PVA NPs corresponds to C═C stretching mode. In addition, the band at 1655cm-.sup.1 assigned to C═O group which could be contributed to the intra/inter molecular hydrogen bond with adjacent hydroxyl group. Moreover, both FT-IR spectra displayed a band at 1095 cm.sup.−1 for pure PVA and 1093 cm.sup.−1 for core-shell Ag/PVA NPs, which are assigned to C—O stretching of acetyl group on PVA back bone. Additionally, the band at 1439 cm.sup.−1 and 1384cm.sup.−1 for pure PVA may be attributed to CH.sub.2 and O—H bending vibrations. However, these bands underwent a blue shift and appeared at 1424 cm.sup.−1 and 1332 cm.sup.−1 in the spectrum of core-shell Ag/PVA NPs. This blue shift of the bands observed at 1424 cm.sup.−1 indicates that PVA polymer is adsorbed on the surface of AgNPs via the interaction of AgNPs and OH groups of PVA.

[0034] FIG. 12 shows according to an exemplary embodiment of the invention FTIR spectra of Pure PEG and core-shell Ag/PEG NPs. The FT-IR spectra of pure PEG and core-shell Ag/PEG NPs, also confirmed the formation of core-shell Ag/PEG NPs. The FT-IR spectra of pure PEG and core-shell Ag/PEG NPs are nearly identical. The FT-IR spectrum of pure PEG shows a band at 3451 cm.sup.−1 corresponds to O—H stretching vibrations and a band at 2888 cm.sup.−1 corresponds to C—H stretching vibrations. Both FT-IR spectra showed bands at 1466 and 1342 cm.sup.−1, which are assigned to C—H bending vibrations. The stretching vibrations of alcoholic OH and C—O—H stretching are observed at 1281 and 1099 cm.sup.−1 also appear in both spectra. However, the FT-IR spectrum of Ag/PEG NPs showed a broad band at 1099 cm.sup.−1 as compared to FT-IR spectrum of PEG, which could be ascribed to C—O—H vibrations of AgNPs in PEG. Comparing the FT-IR of both pure PEG and the spectrum of core-shell Ag/PEG NPs, a strong band is observed at 509 cm.sup.−1 at the spectrum of core-shell Ag/PEG NPs, which could be attributed to AgNPs banding with oxygen from hydroxyl groups of PEG chains and thus suggesting the existence of van der Waals interaction between the positively charged groups on the surface of Ag NPs and the negatively charged oxygen from the hydroxyl groups of PEG.

[0035] FIG. 13 shows according to an exemplary embodiment of the invention FTIR spectra of Pure PVP and core-shell Ag/PVP NPs. The FT-IR spectra of pure PVP and core-shell Ag/PVP NPs formed by polyol method shown are closely identical. Both spectra showed peaks at 3430 cm.sup.−1 which corresponds to O—H stretching vibration of hydroxyl group and 2952 cm.sup.−1 corresponding to symmetric stretching vibration of C—H bond. Both spectra also showed a sharp band at 1655 cm.sup.−1 that corresponds to amide carbonyl stretch absorption and peaks at 1463, 1439, 1424 cm.sup.−1 are assigned to the vibration of tertiary nitrogen. However, the FT-IR spectrum of core-shell Ag/PVP NPs displays a red shift at band 1099 cm.sup.−1, compared to the band at 1075 cm.sup.−1 in the spectrum of pure PVP. The red shift of band at 1099 cm.sup.−1 confirms the involvement of pyrrolidyl nitrogen electrons in the formation of core-shell Ag/PVP NPs. This emphasizes that PVP is adsorbed at the silver NPs surfaces through donating electrons from the N atom to the Ag or the coordination between N and Ag. Wang et al. revealed that core-shell Ag/PVP NPs with diameters smaller than 50 nm are protected by PVP via a coordination bond between N in PVP and Ag as shown in while NPs with diameters greater than 50 nm both N and O in PVP coordinated with Ag.

DETAILED DESCRIPTION

[0036] 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'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's dose, and (6) DOX's toxicity.

[0037] Method

[0038] The NPs-based combinatorial therapy according to an embodiment of the present invention (FIG. 1) can be been prepared as follows:

[0039] (1) Preparation of silver nanoparticles coated with FDA-approved synthetic polymers (PVA, PEG and PVP),

[0040] (2) Drug loading (DOX), and

[0041] (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.

[0042] 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:

[0043] (1) Testing the cytotoxic effect of different concentrations (2, 4, 8, 10, and 12 μg/ml) of free DOX alone,

[0044] (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

[0045] (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).

[0046] A Prior Art Example to Contrast the Embodiments of the Present Invention

[0047] 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:

[0048] (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),

[0049] (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

[0050] (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.sub.50 were measured following 48 hrs incubation [20].

[0051] In-Vitro MTT Results

[0052] The Cytotoxic Effect of AgNPs

[0053] After 48 hrs incubation, the cell viability was measured and the results were plotted in graphs (FIGS. 2A-D). From these graphs, IC.sub.50 were determined to be 48 μg/ml for AgNPs and >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.sup.+ ions. The released Ag.sup.+ 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].

[0054] 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' surface coating controls AgNPs' dissolution, which is directly correlated with their cytotoxicity [22-24]. Results revealed that Ag/PVP NPs exhibited the highest cytotoxicity (IC.sub.50: 42 μg/ml) as compared to AgNPs (IC.sub.50: 48 μg/ml), core-shell Ag/polymeric (PVA and PEG) NPs (IC.sub.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.sup.+ 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.sup.+ ions, hence reducing Ag.sup.+ to Ag.sup.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.sup.− ions, forming stable Ag-ligand complexes resulting in Ag.sup.+ 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.sub.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.

TABLE-US-00001 TABLE 1 Comparison between IC50 of AgNPs in the prior art (Hekmat et al.) and the invented Ag/polymeric NPs on breast cancer cell lines (MCF-7). Prior Art Hekmat et al. The invented Ag/polymeric NPs P.O.C AgNPs Ag/PVA NPs Ag/PEG NPs Ag/PVP NPs IC.sub.50 9.35 μg/ml >100 μg/ml >100 μg/ml 48 μg/ml

[0055] The Cytotoxic Effect of NPs-Based Combination Therapy (According to this Invention) and Conventional Combination Therapy (Present in the Prior Art)

[0056] 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 (FIGS. 3A-E), the IC.sub.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.sub.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.sub.50 at 10-fold reduced doses compared to free DOX alone. In addition to the low concentration of DOX, the IC.sub.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].

[0057] On the other hand, the in-vitro results obtained by Hekmat et al. showed that the IC.sub.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.sub.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.sub.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.

[0058] 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.

[0059] 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.

TABLE-US-00002 TABLE 2 IC50 of the NPs-based combination therapy according to the invention compared to the DOX-Ag combination in the prior art. The NPs-based combination therapy Prior Art according to the invention Hekmat et al. DOX-Ag/ DOX-Ag/ DOX-Ag/ P.O.C DOX + AgNPs PVA NCs PEG NCs PVP NCs IC50 0.23 μg/ml 0.19 μg/ml 0.14 μg/ml 0.1 μg/ml DOX + DOX DOX − DOX 2.55 μg/ml 3.4 μg/ml 3 μg/ml 3.5 μg/ml AgNPs Ag/PVA Ag/PEG Ag/PVP NCs NCs NCs % of 18% 40% 57% reduction in DOX dose

[0060] Additional Results

[0061] Synthesis and Characterization of AgNPs and Core-Shell NPs

[0062] 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 (FIG. 4A-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 (FIGS. 4B, 4D, 4F, and 4H), respectively. The UV-Vis spectra of AgNPs, Ag/PVA NPs and Ag/PEG NPs (FIGS. 4A-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 (FIG. 4D) 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 FIGS. 10-13.

[0063] Synthesis and Characterization of DOX-NCs

[0064] 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 (FIGS. 5A-D) and SEM (FIGS. 6A-H). The UV-Vis spectra (FIGS. 5A-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.

[0065] In-Vitro Drug Release

[0066] 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 (FIG. 6A-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 (FIG. 7A) and PBS pH 7.4 (FIG. 7B).

[0067] In-Vitro Cytotoxicity Assay

[0068] Effect of AgNPs and Core-Shell Ag/polymeric on MCF-7 Cells and 1BR hTERT Cells

[0069] 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 (FIGS. 8A-D) in a dose-dependent manner. The inhibitory concentration (IC.sub.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.sub.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 (FIGS. 8A-D).

[0070] Effect of DOX-Core-Shell Ag/Polymeric NPs on MCF-7 Cells and 1BR hTERT Cells

[0071] 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.sub.50 of free DOX on MCF-7 cells was determined to be 3.7 μg/mL (FIG. 14). Based on the IC.sub.50 of free DOX, lower DOX-NCs concentrations than the calculated IC.sub.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.sub.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 (FIGS. 9A-D). On the other hand, the estimated IC.sub.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 (FIGS. 9A-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.

[0072] Conclusion

[0073] 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.

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