WO2018069896A1

WO2018069896A1 - Drug conjugated ultra-small gold nanoparticle for effective killing of drug resistant cancer cells

Info

Publication number: WO2018069896A1
Application number: PCT/IB2017/056384
Authority: WO
Inventors: Aleem Ahmed DR. KHAN; Sandeep Kumar DR. VISHWAKARMA; Avinash DR. BARDIA; Syed Ameer Basha DR. PASPALA; Habeeb DR. MD. AEJAZ
Original assignee: Dr Khan Aleem Ahmed
Priority date: 2016-10-15
Filing date: 2017-10-14
Publication date: 2018-04-19

Abstract

The invention described herein is to produce ultra-small size nanoconjugates of anti-cancer drugs for the reversal of drug resistance mechanisms in cancer cells. Nanoconjugates developed herein comprised of Sorafenib (SF, an anti-cancer drug) conjugated with biocompatible, ultra-small size gold nanoparticles (GNPs). Also, provided the method for nanoformulation of SF-GNP nanoconjugates and its potential to combat the drug resistance in human hepatoblastoma cells.

Description

DRUG CONJUGATED ULTRA-SMALL GOLD

NANOPARTICLE FOR EFFECTIVE KILLING OF DRUG RESISTANT CANCER CELLS

Technical field of the invention

[001] The present invention relates with the development of ultra-small size and biocompatible sorafenib-gold (SF-GNP) nanoconjugates for the treatment of drug resistant cancer cells. The nanoformulation of SF-GNP was obtained using colloidal suspension of GNPs with SF through electro-static interaction. The said nanoconjugate was characterized using different methods to investigate their interaction with the cells and resistance mechanisms in cancer cells. Both ex vivo and in vivo models were used to identify the biological compatibility of SF-GNP nanoconjugates at different levels. The efficacy of said nanoconjugate in SF- resistant cancer cells was evaluated at cellular and molecular levels to reveal the major mechanisms for combating the drug resistance.

Brief summary

[002] This invention relates with the development of ultra-small size, stable and biocompatible nanoconjugates of anti-cancer drugs. These nanoconjugates are highly stable as it does not react with oxygen, water or serum. These nanoconjugates were synthesized using conjugation of highly pure and ultra-small size gold nanoparticles (GNPs) with anti-cancer drug (Sorafenib). The conjugation of sorafenib with GNPs was generated by electrostatic interaction in aqueous colloidal suspension resulting in SF-GNP nanoconjugates referred herein. SF- GNP nanoconjugate mediates enhanced chemotherapeutic response to combat drug resistance in cancer cells.These SF-GNP nanoconjugates were able to evade drug resistance in cancer cells.

Background of the invention

[003] Drug resistance in cancer, particularly in hepatocellular carcinoma is a major delimiting factor in treatment (Jemal et al. 2011, Torre et al. 2012). Despite the availability of a wide range of therapeutic molecules with different molecular structures and cellular targets, an overall increase in drug resistance has been observed in cancer cells (Holohan et al. 2013). Combination treatment strategy using multiple drugs has also failed to cure the cancer drug resistance. Continuous exposure to high dose of anti-cancer drugs may also lead to certain fatal mutations in healthy tissues that result in several adverse effects in the body. There is no effective treatment option for cancer drug resistance and a huge number of deaths are occurring due to continuous treatment failure in drug resistant cancer patients. Hence, treatment of drug resistant cancers has been a major challenge because it is difficult to lower the drug doses with high efficiency without causing much adverse effects and avoid the drug resistance mechanisms.

[004] The emergence of nanotechnology holds great potential in diagnosis, prognosis and most importantly in theranostic application for cancer treatment. Since last decade, a variety of nanoparticles have been prepared using various novel strategies (WO2012018383A2, US8673358 B2, Chen et al. 2007). However, each method has limitation of synthesizing biologically safe nanoparticles which can be used most frequently for delivering therapeutic molecules into human body. The toxicity of the nanoparticles attributed to the size, dose, surface chemistry, side chains and the type of stabilizing agent used (Zhang et al. 2011, Vecchio et al. 2012, Fraga et al. 2013). In consideration of these points, synthesis of ultra-small and highly stable gold nanoparticles was carried out due to its unique inert property, ease of surface functionalization and accumulation at inflammatory and tumor sites.

[005] Sorafenib is one of the most effective drugs for the treatment of hepatocellular carcinoma, renal and other cancers. However, the decisive side effects of sorafenib, limits its continuous dose to the cancer patient. In addition, cancer patients having resistance for sorafenib do not have secondary option for continuous treatment (Bruix et al. 2011). Thus for unravelling the mechanisms to treat drug resistant cancer cells, an attempt was made to aid the newer therapeutic strategy against sorafenib resistant human hepatoblastoma cells. Wherein, the current invention provides a novel strategy for the treatment of drug resistant cancer cells using the same drug at low dose. This invention includes synthesis of highly safe ultra-small gold nanoparticles which can be conjugated with sorafenib and could be more effective in killing drug resistant cancer cells. The reason to synthesize such metal nanoparticle was due to its excellent conductive properties, high biocompatibility and stability as it does not react with water/and or oxygen, enhanced permeability and retention effect in cancer tissues and imminent carriers for effective cellular delivery of diverse bioactive molecules and drugs. These properties make these metal nanoparticles very practical to preclinical and clinical uses.

[006] This invention describes a rapid and facile synthesis method for preparing gold nanoparticles from AuC13 precursor. These nanoparticles are stable in aqueous solution without the use of any additional stabilizing agent for the capping. Further nanoformulation of sorafenib with GNPs was performed using GNP-FITC nanoprobe and termed as sorafenib-gold nanoparicle (SF-GNP) conjugate. Biological safety of SF-GNP nanoconjugates was identified in vitro on human hepatocytes for cell viability and proliferation at different time points and concentrations. Animal models were used to test the in vivo safety of the nanoparticles and GNPs tagged with sorafenib using biochemical parameters and histological evaluation of the vital organs. The long-term exposure of these nanoparticles didn't show any adverse effects on cellular and molecular dynamics of the biological system both in vitro and in vivo.

[007] Further the therapeutic efficacy of SF-GNP was evaluated in vitro on SF resistant human hepatoblastoma cell line (HepG2) at different concentrations with time. SF-GNP nanoconjugates showed killing of SF resistant HepG2 cells in solid tumor model system. The molecular investigation was performed to identify the down-regulation of key molecular pathways (such as ABC transporters, TGF- β, hURP and others) involved in cancer development and progression (Li et al. 2016). In summary, the present invention provides an opportunity for safe delivery of drugs and other molecules in conjugation with GNPs to reverse drug resistance in cancer cells. The unique strategy adopted in this invention of preparing nanoformulations of anti-cancer drugs have potential to overcome drug efflux mechanisms and delivery barriers in solid tumors which will provide new hope in the treatment of cancer drug resistance.

Brief description of drawing

[008] Fig. 1 Schematic representation showing preparation of GNPs using AuCB as precursor by LiBH4 reduction process. Further the prepared GNPs are functionalized with FITC to generate GNP-FITC nanoprobe which is used to formulate SF-GNP nanoconjugates

[009] Fig. 2 (A) UV-vis spectra of GNPs showing strong Surface Plasmon Resonance (SPR) peak at 524nm and optical image of GNP colloidal aqueous solution (B) Transmission electron microscopy (TEM) image of synthesized ultra- small GNPs showing size <10nm (C) UV-vis spectra showing differential peaks of FITC, FITC-GNP, SF and SF-FITC-GNP (D) Fluorescence resonance energy transfer (FRET) intensity plots showing quantification of FRET process for FITC and GNP at various concentrations © Fluorescence spectra showing peaks of FITC, FITC-GNP and SF-FITC-GNP (F) TEM image of SF-GNP nanoconjugates representing the ultra-small size of <10nm

[010] Fig. 4 Histological analysis of liver, kidney, lung, heart and brain tissues using hematoxyleine and eosine staining at (A) day 3 and (B) day 14 post- intraperitoneal injection of GNP, SF and SF-GNP nanoconjugates in male Wister rats. Pathological investigations revealed absence of any abnormal or pathological signs such as tissue inflammation, hemorrhage, fibrotic response, nuclear centralization, changes in the organization of ECM and cellular components or deposition of any adjacent fatty tissue which represents biologically safe nature of prepared SF-GNP nanoconjugates and GNPs for future biological applications (Scale bar: 20μπι, Magnification: 40X)

[011] Fig. 5 (A-F) Analysis of early and late immunological response by quantifying inflammatory markers IL-6 and IL-Ιβ at day 3, day 7 and day 14 did not show change in their levels representing that intraperitoneal exposure of GNP and SF-GNP nanoconjugates do not initiate immunological reactions (G) Estimation of renal function test (RFT) such as urea and creatinine in serum samples of rats post-intraperitoneal injection of GNP and SF-GNP showed no significant change (H) Similarly, liver function tests (LFT) did not reveal significant variation in serum levels of biochemical parameters

[012] Fig. 6 Body weight profile of animals in different groups did not show significant change for 14 days after intraperitoenal administration of GNP and SF- GNP nanoconjugates

[013] Fig. 7 Serum stability analysis of SF-GNP nanoconjugate did not show any significant change under the effect of variable concentrations of serum addition

[014] Fig. 8 Microscopic analysis of SF-resistant solid tumor cell colonies before and after treatment with SF-GNP nanoconjugates for different time points. The analysis revealed that treatment with SF-GNP nanoconjugates reduces the colony size and increases the deformalities in cellular packaging inside the colonies with increasing the time of exposure (Scale bar: 20μπι, Magnification: 40X)

[015] Fig. 9 Colony formation assays in drug resistant HepG2 cells revealed significant decrease in percentage cell survival along with reduction in colony number and size with increase in time of exposure to SF-GNP nanoconjugates from day 1 to day 14. Other treatment groups (SF and GNP) didn't show significant change at any time point revealing that SF-GNP nanoconjugates has intrinsic property to overcome drug resistance in cancer cells

[016] Fig. 10 Heat map showing the gene expression levels in control ©, SF- resistant (SR-C), SF resistant cells treated with GNP (SR-A) and SF resistant cells treated with SF-GNP nanoconjugates at day 3, day 7 and day 14 post-treatment. Relative quantification of major cancer pathway transcripts (hURP and TGF-β), Warburg oncogene (CD 147) and drug transporter (ABCG2) showed significant downregulation after treatment with SF-GNP nanoconjugates with increasing the treatment duration. The downregulated expression of cancer pathway genes showed molecular level control over the SF resistance and more specifically decrease in ABCG2 expression provided evidence for the inhibition of drug efflux mechanisms which is the major cause of drug resistance by preventing drug uptake

Fig. 11 Microscopic analysis showing dose dependent reduction in the development of SF resistant HepG2 cell colonies after treatment with SF-GNP nanoconjugates in dose dependent manner

[017] Fig. 12 (a-c) Cell survival assay in SF resistant HepG2 cell colonies showed that post-treatment with DF-GNP nanoconjugates significantly reduces the percentage cell survival with increasing the time from day 3 to day 14 (d) Heat map showing the changes in gene expression levels at day 3, 7 and 14 after treatment with SF-GNP nanoconjugates to SF resistant HepG2 cells with single, double and triple dose. No much significant difference was observed during treatment of different doses at any time point (p>0.05)

[018] Fig. 13 Schematic representation showing the molecular mechanism through which SF-GNP nanoconjugate contributes to additional enhanced chemotherapeutic response in SF resistant HepG2 cells (a) Common mechanism of SF resistance in cancer cells showing activation of membrane transport molecules which block the entry of SF molecules and efflux the overdose or continuous exposure of high dose of SF from the cells (b, c) Proposed mechanism for SF-GNP entry into SF resistant cancer cells by evading membrane transporters resulting in reduced expression of ABCG2 which is not sufficient to block the entry of SF-GNP into the cells. In addition, the low dose of SF-GNP may also have one reason not to affect the ABCG2 which does not produce early SF resistance in cancer cells. Further, the down regulation of hURP may result in disrupted spindle formation during cancer cell division which could inhibit the proliferation of SF resistant HepG2 cells after treatment with SF-GNP. The declined expression of TGF-β transcript expression after SF-GNP treatment may decrease the EMT ultimately resulting in declined chemoresistance and immunocompetency of SF resistant HepG2 cells. Further, the decrease in the expression of CD 147 results in enhanced MMPs production which may degrade the extracellular matrix (ECM) of cancer cells and decrease the proliferation of SF resistance HepG2 cells after treatment with SF-GNP conjugate.

Detailed description of the invention

[019] The invention provides highly stable, biologically compatible, ultra-small size gold nanoconjugates of sorafenib. The nanoconjugate described herein involved the complete reduction of the salt used for preparation of gold nanoparticles as described in example 1. Thereafter, nanoconjugate was characterized and used to treat sorafenib-resistant human hepatoblastoma cells in ex vivo solid tumor model system as described in forecoming examples of each embodiment. The cellular and molecular level analysis was performed to evaluate the mechanisms of drug resistance against the nanoconjugates. Example 1

Preparation and characterization of ultra-small size GNPs

[020] Ultra-small size gold nanoparticles (GNPs) were synthesized by instantreduction of Gold chloride (AuC13, 0.2 mM) with 6 mM of Lithium Borohydrate (LiBH4) at room temperature. The stabilization agents were not used which provides highly pure GNPs free from organic capping. This method is slightly modified rapid and facile method of earlier invention (WO2015063794A3) to generate salt free nanoparticles. Herein the invention describes a method of preparing GNPs after complete reduction of LiBH4 at room temperature. The GNPs synthesized in aqueous medium herein shows a strong Surface Plasmon Resonance (SPR) peak at 524nm in UV-vis absorption spectra. Further transmission electron microscopy (TEM) analysis revealed average size of approximately 7nm particle size which was further confirmed by hydrodynamic radius measurements.

Example 2

Formulation of SF-GNP nanoconjugates

[021] The nanoformulation of SF with GNP was optimized by first preparing a conjugate of GNP with fluorescein isothiocynate (FITC) which was termed as fluorescence quenched nanoprobe. The confirmation of formulation of SF-GNP nanoconjugates was performed by estimating the relative decrease in fluorescence peak intensity of FITC in colloidal suspension of GNPs. Due to the electrostatic interaction, fluorescence resonance energy transfer (FRET) was optimized between FITC and GNPs. The absence of characteristic peak of FITC at lower concentration was taken in consideration to confirms the functionalization of - NCS group of FITC on the surface of GNPs. This FITC-GNP nanoprobe was used to formulate SF-GNP nanoconjugate by replacing FITC with SF. SF-GNP conjugation efficiency was determined by the amount of reappearance of FITC fluorescence in aqueous medium. To generate the nanoformulation of SF with GNP different concentrations of SF (2-20C^g) in 3 millilitres of colloidal suspension having 0.667μ1 of FITC which is functionalized on GNPs. The formulation of SF-GNP nanoconjugate was determined by relative increase in FITC fluorescence from FITC-GNP nanoprobe with addition of increased SF which is an indicative of release of FITC from FITC-GNP nanoprobe and binding of SF on GNP. The optimum condition for the conjugation of more than 80% of SF on GNP was optimized by adding 0.33mg/mL stock solution of SF in 1 :2 ratio with GNPs. Confirmation of SF-GNP nanoconjugate was identified with the reappearance of fluorescence spectra with the increase in SF concentration and gradual reduction in FITC fluorescence in FITC-GNP nanoprobe. The estimated size of the SF-GNP nanoconjugate was identified to be approximately 8nm using TEM imaging which was further confirmed by dynamic light scattering measurement and HPLC analysis. In HPLC, the percentage loading of SF on GNPs was estimated by the relative decrease in the area of free SF in relation to the area found in the same concentration of SF-GNP nanoconjugate prepared in aqueous colloidal suspension medium. Measurement of zeta potential of SF-GNP nanoconjugate revealed ~63mV with slight reduction in negative potential with reference to GNP (~43mV) and free SF (~3mV). This measurement has vital significance in drug uptake mechanisms within the cellular system.

Example 3

Identifying in vitro toxicity of SF-GNP nanoconjugates

[022] In vitro toxicological response of cells against SF-GNP nanoconjugates was determined by exposing human hepatocytes with SF-GNP nanoconjugate for different time pints. In vitro toxicity studies on primary human hepatocytes was performed up to 72 hours using MTT cell viability assay. Simultaneously LDH assay was also performed to identify the cell membrane damage during exposure to SF-GNP nanoconjugates. Five different concentrations of SF-GNP nanoconjugates (1, 1/10, 1/100, 1/1000 and 1/10000) were used during these investigations. These investigations were performed on matured human hepatocytes with 70-80% confluency representing 3-4 million cells/well in culture plates. Each condition was established in triplicates and conducted at least five times to replicate the results. The in vitro cytocompatibility of SF-GNP nanoconjugates demonstrated no change in optical densities at any time points measured by spectrophotometric analysis. This finding revealed that ultra-small size SF-GNP nanoconjugates are non-toxic to the healthy human primary hepatocytes.

Example 4

Identifying in vivo response of SF-GNP nanoconjugates

[023] Over the past decades, the pre-clinical and clinical applicability of nano- delivery systems has been limited due to the potential risk to the biological systems. Hence, one of the embodiments of the present invention was also to identify the biological safety of SF-GNP nanoconjugates. Male Wistar Rats (average body weight 140 ± 25 g were) were used to evaluate the in vivo response for SF-GNP nanoconjugates. To evaluate the toxicological responses, different doses of SF-GNP nanoconjugates at different concentrations were injected intraperitoneally. The motivation for intra-peritoneal administration of SF-GNP nanoconjugate was that the peritoneum is highly rich 21vascularised immunologically privileged site, it does not produce higher immunological response immediately due to absence of large number of immunological cells as compared to the other sites. In addition, these nanoconjugates are very small (<10nm), hence can easily enter through endothelial barrier of the blood vessel in peritoneum (>100nm in size) which leads their direct accumulation into the liver through gastro-intestinal tract. This site also has advantages over rescue of the reticulo-endothelial uptake by immunological cells which is a positive sign for enhanced delivery at the targeted cells. [024] The does for SF in animal experiments were determined with the standard dose used for treatment in human (approximately 6.66mg/kg body weight). In our experiments we have reduced one third of this dose concentration. This is because while preparing SF-GNP, the maximum loading capacity of SF on the GNP was about 80% of 1.11 mg/mL, hence we have selected 2.22 mg/kg as a maximum dose which did not showed significant toxicity therefore no need to perform experiments with lower doses. The biochemical analysis revealed normal level of serum biomarkers specific to the liver and renal functions. In addition to this, haematological investigations also showed no change in complete blood profile. Histological analysis of vital organs such as liver, kidney, heart, lungs and brain did not show evidence of inflammatory reactions, haemorrhage, cellular necrosis, tissue disorganization or fibrotic responses.

Example 5

Assessment of serum stability of SF-GNP nanoconjugates

[025] Serum stability is one of the most crucial factors while considering the preclinical and clinical applicability of SF-GNP nanoconjugates. Therefore, the serum stability was identified by incubating four different concentrations of SF- GNP nanoconjugates (1, 1/16.66, 1/25, 1/50) with human serum for 30min. The analysis revealed no change in absorbance peaks of SF-GNP nanoconjugates after incubation with serum. Example 6

Establishing ex vivo drug-resistant solid tumor model system

[026] For the assessment of SF-GNP nanoconjugate efficacy in drug resistant cancer cells, IC50 value of SF was determined though MTT by culturing human hepatoblastoma cells (HepG2) in soft agar medium. This three-dimensional culture model system was used to have better correlation between ex vivo transformation and in vivo tumorigenesis. The SF resistant cells were established by increasing concentration of SF (25% each time) above the IC50. The optimal concentration obtained to develop resistance in HepG2 cells was determined and used to evaluate the resistance index for both free SF and SF-GNP nanoconjugates.

Example 7

Treatment of drug-resistant cancer cells using SF-GNP nanoconjugates

[027] The killing efficiency of SF-GNP nanoconjugates in SF resistant HepG2 cells in solid tumor model system was determined at day 3, 7 and 14 post- treatment at three different concentrations (1, 1/100 and 1/1000) at single, double and triple doses. The parameters for this investigation involved assessment of colony size, colony number, percentage cell survival and cancer transcriptome analysis (hepatoma upregulated protein: hURP, CD 147 and TGF-β) including drug transporter which is one of the essential molecular regulator for drug resistance in cancer cells. These investigations revealed significant reduction in colony numbers, size and percentage survival of SF resistant cells after treatment with SF-GNP nanoconjugates. In addition to this, significant downregulation in cancer related gene transcripts showed activity of SF-GNP nanoconjugates to combat the SF resistant cancer cells. More importantly, significant reduced level of ABCG2 expression estimated by mRNA and protein levels analysis revealed that SF-GNP nanoconjugates evade the drug efflux mechanism in resistant HepG2 cells which allows sufficient amount of accumulation of SF-GNP nanoconjugates required for efficient killing.

[028] References

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Claims

Claims:

1. A method of preparing ultra-small size sorafenib-gold (SF-GNP) nanoconjugates (<10nm) in aqueous colloidal medium.

2. The method of claim 1, wherein for nanoformulation of SF-GNP nanoconjugates no capping or stabilizing agents were used.

3. The nanoformulation of claim 1 wherein the said nanoconjugate is biologically compatible.

4. The nanoconjugate of claim 1 has high stability in serum.

5. The nanoconjugate of claim 1 has property to fight with drug-resistant cancer cells wherein it reduced the cell survival and formation of solid tumor colonies.

6. The SF-GNP nanoconjugate of claim 1, has dose and time dependent effect on the killing of drug-resistant cancer cells in solid tumor colonies.

7. SF-GNP nanoconjugates of claim 1, downregulates the expression of cancer pathway genes and ameliorates the death of resistant cancer cells.

8. SF-GNP nanoconjugates of claim 1, downregulates expression levels of drug transporter transcript (ABCG2) which results in high drug uptake inside the resistant cancer cells thereby reducing the drug efflux mechanisms.

9. The method of claim 1 wherein sorafenib may be replaced with other anticancer drugs to prepare nanoformulation with GNPs used herein to combat the drug resistance in other type of cancer cells.