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Paclitaxel nanocrystalline assemblies as a potential transcatheter arterial chemoembolization (TACE) candidate for unresectable hepatocellular carcinoma
Materials Science & Engineering C 107 (2020) 110315
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Paclitaxel nanocrystalline assemblies as a potential transcatheter arterial chemoembolization (TACE) candidate for unresectable hepatocellular carcinoma
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Atul Dev, Ankur Sood, Subhasree Roy Choudhury, Surajit Karmakar∗ Institute of Nano Science and Technology, Mohali, Punjab, India
A B S T R A C T
We have prepared a water dispersible Nanocrystalline hierarchical structure of paclitaxel using self-assembly approach. Its unique structure provides the advantage of increased payload and higher retention of a chemotherapeutic drug inside the tumor site for a prolonged period. Micron scale structure, sustained release behaviour, multichannel fluorescence property and devoid of any vehicle molecule makes it promising in treating solid tumors through localized drug delivery approach like TACE. This method is based on one pot synthesis through co-precipitation reaction followed by crosslinking. Therapeutic efficacy of the molecules is successfully verified through the significant reduction in the volume of the 3D spheroid model of hepatocellular carcinoma (HCC).
1. Introduction Hepatocellular carcinoma (HCC) is the primary form of Liver cancer that originates in the liver. It is the third leading cause of cancer-related deaths and represents 70–85% cases of primary liver cancers worldwide [1]. Despite therapeutic advances, liver cancer remains one of the most challenging forms of cancers to treat. Surgical resection is the primary treatment for BCLC (Barcelona clinic liver cancer) stage A patients characterized by solitary lesion > 2 cm or up to 3 multifocal lesions measuring less than 3 cm. Sorafenib, a multiple kinase inhibitor is the first line therapy to treat BCLC stage C patients who have reduced hepatic functions with vascular invasion or distant metastasis. Patients, who fail Sorafenib treatment has shown significant improvement in survival using Regorafenib [2]. Although, development of second line systemic therapies after Sorafenib is still in early stages. Lack of selectivity, recurrence, multidrug resistance and severe side effects of systemic chemotherapy significantly limits the success rate in the treatment of HCC. Therefore, it is of great clinical importance to develop new strategies to overcome these issues. Local drug delivery approaches could provide benefits of minimal systemic toxicity, an increased payload of the drug with attenuation of multidrug resistance (MDR) [3]. Of lately, Transcatheter arterial chemoembolization (TACE) has become the most common approach for the management of HCC in BCLC stage B patients. It is often recommended for BCLC stage C patients along with Sorafenib treatment to further improve the survival. It combines the injection of chemotherapeutic drug with simultaneous
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occlusion of the tumor blood supply. HCC exhibits intense neoangiogenic activity and is mostly dependent on the hepatic artery for blood supply, while the rest of the liver is supplied by the portal vein [4]. This provides the rationale to use arterial obstruction as an effective therapeutic option, as it induces ischemic tumor necrosis. TACE showed increased survival rate in comparison to other best supportive care [5–9]. Despite being the effective treatment method, TACE suffers from some serious drawbacks such as generation of hypoxia condition during embolization which in turn leads to aggressive phenotypes of tumor with increased angiogenesis and metastasis activity [10]. The chemoresistance and recurrence of HCC are other critical problems associated with tumor hypoxia [11,12]. As a result of this, complete neutralization of tumor cells is not possible by chemotherapeutic drugs being used in TACE. To achieve significant survival in TACE, improvement in current approaches is immensely required. Effective killing along with negligible generation of hypoxia condition is the prerequisite for a formulation to be notably effective for its utilization in TACE. Currently, the most common anticancer drug used in TACE is doxorubicin (36%), followed by cisplatin (31%), epirubicin/doxorubicin (12%), mitoxantrone (8%), mitomycin C (8%) and a chemical conjugate of a synthetic copolymer of styrene maleic acid (SMA) and the proteinaceous anti-cancer agent neocarzinostatin (NCS) SMANCS (5%) [8]. Interestingly, Paclitaxel (PTX) which is one of the most effective drugs against lung, ovarian, and breast cancer [13] has not been frequently used in TACE owing to its current commercial formulation available which includes organic solvents polyoxyethylated castor oil
Corresponding author. Institute of Nano Science and Technology, Habitat Centre, Phase 10, Sector 64, Sahibzada Ajit Singh Nagar, Punjab, India. E-mail address: [email protected] (S. Karmakar).
https://doi.org/10.1016/j.msec.2019.110315 Received 30 January 2019; Received in revised form 1 October 2019; Accepted 14 October 2019 Available online 17 October 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
Materials Science & Engineering C 107 (2020) 110315
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at 10000 g for 30min and was washed three times using double distilled water and lyophilized for further use.
(Cremophor EL) and dehydrated ethanol (1:1, v/v). Theses organic contents are reported to induce severe toxic effects which include neurotoxicity, hypersensitivity, cardio-toxicity, anaphylaxis and nephrotoxicity [14]. Thus various alternative nano platforms like drug nanocrystals [15], polymers [16], lipids, drug polymer conjugates, magnetic nanoparticles [17], inorganic nanoparticles etc. were investigated for the delivery of PTX in different cancer models including HCC. Despite improved attributes of nano-delivery systems, systemic clearance and non-specific distribution in organs still impede the clinical usage of nano-formulations in TACE [18]. This work reports the synthesis of a novel structural assembly of paclitaxel nanocrystals, which could be directly introduced into the tumor site with the help of a catheter. Drug nanocrystals gained interest in delivering poorly water-soluble anticancer drugs due to the high drug loading efficiency, circulation stability [19] and ability to resolve multidrug resistance [20]. The prominent advantage of nanocrystals is the absence of delivery vehicle that may introduce adverse or unwanted toxic reactions into the human body. The current approach well suits for PTX to produce Cremophor EL®-free formulations. Several studies on PTX nanocrystals have been carried out for the treatment of various cancer [21,22] which signify its therapeutic efficacy. The unique structural arrangements of PTX nanocrystals could permit improved chemotherapeutic effect through increased payload of the drug and simultaneous occlusion of the blood flow with minimum hypoxia effect. This structural assembly could find its utilization in TACE therapy as a potential chemotherapeutic agent. The current work addresses the challenges of systemic delivery and TACE procedures. We have reported a highly fluorescent, water dispersible, nanocrystalline assemblies of PTX drug which shows sustained drug release profile as compared to the bare PTX molecule. Inbuilt multichannel fluorescent properties make these particles expedient during surgical procedures, the ability of real-time monitoring of such particles in the course of catheter mediated delivery could provide edge over currently available formulations. These PTX astral assemblies could provide structural advantage over currently available TACE formulations. Its unique astral appearance promises prolonged retention inside tumor with minimum passive diffusion to the nearby healthy tissues. Current hierarchical self-assembled PTX structures closely resemble with earlier reported structures [23] but are unique in its ease of one pot synthesis, morphology and inherent fluorescence property. One pot synthesis of such structures allows structural uniformity and ease of scaleup during large scale production.
3.2. Characterization X-ray diffractograms (XRD) were recorded on a Bruker powder XRD D8 X-ray diffractometer over 2θ range of 5°–40° using Cu Kα X-rays of wavelength1.54 Å operated at 30 mA & 40 kV. Scanning electron microscopy (SEM) images were obtained with JEOL at 10–20 kV. Fourier transform infrared (FTIR) spectra were recorded on Cary Agilent 660 IR spectrophotometer by mixing the samples with potassium bromide and making pellets for analysis. For each spectrum, 256 scans and 4 cm−1 resolution were applied over the range of 400–4000 cm−1. UV-Visible spectrophotometer Shimadzu UV-2600 was used to estimate the drug release. Fluorescence images were captured using Zeiss confocal laser scanning microscope. 3.3. 1H NMR studies Proton (1H)-NMR studies were carried out for the analysis of anhydrous PTX (P1), reaction intermediates (P2 and P3) and PTX astral assemblies (P4) respectively. Samples to be investigated were dissolved in D-6 DMSO and analysed using Bruker Av III HD at 400 MHz and 25 °C. The chemical shift values are reported in ppm scale using tetramethylsilane (TMS) as an internal standard. 3.4. In-vitro drug release studies In vitro drug release studies were performed in PBS pH 7.4 using 12KD cut-off dialysis membrane. One mg of PTX and PTX nano-assemblies were weighed separately and suspended in 1 ml of PBS solution containing 0.1% of SLS. Drug release was analysed in 20 ml of PBS solution as a releasing media at 37 °C with stirring at 150 rpm. At different time intervals one ml of aliquot was taken, and sink was replaced with the same amount of solvent, aliquot was analysed using UV–Vis spectrophotometer after preparing standard curve of PTX in methanol at 231 nm. 3.5. Spheroid generation and treatment Spheroid was generated using a hanging drop culture technique. HepG2 cells were cultured in DMEM media supplemented with 10% FBS maintaining 2000 cells per hanging drop. After 7 days of culture at 37 °C and 5% CO2, spheroids were collected for treatment. Spheroids were then treated with a clinically relevant concentration of PTX (10 nM) in form of astral structures and imaged at various time points. Scanning electron microscopy was performed to verify the presence of astral structure inside the spheroid.
2. Materials and methods 2.1. Materials Paclitaxel (PTX) was purchased from Clear synth. Sodium lauryl sulfate (SLS), Ethanol molecular grade was purchased from Merck. Glutaraldehyde 25% w/v solution, Dulbecco's Modified Eagle Medium (DMEM), streptomycin penicillin solution, Fetal Bovine Serum (FBS) and Trypsin EDTA were procured from HI-media. DAPI(4′,6-diamidino2-phenylindole), Glycerol, and 12KD dialysis membrane were purchased from sigma Aldrich. Human liver carcinoma cell line (HepG2) was purchased from National Centre for Cell Science, Pune.
3.6. Scanning electron microscopy of spheroids Hep G2 spheroids after 5-day treatment with PTX nano-assemblies were imaged using Scanning electron microscopy. The treated spheroids were washed 3–4 times with chilled PBS and fixed overnight in paraformaldehyde at 4 °C. After overnight fixation the spheroids were washed three times with chilled PBS and subsequently treated with gradient series of ethanol. The spheroids were broken to expose the internal morphology and presence of PTX astral assemblies. Finally, they were sputter-coated with gold and imaged at 10 kV.
3. Methods 3.1. Synthesis of PTX astral structure
3.7. Confocal laser fluorescence microscopy
Paclitaxel was dissolved in ethanol (2 mg/ml) and added dropwise to the antisolvent (water) while maintaining the solvent and antisolvent ratio (3:7). The system was stirred continuously for 15 min at 1000 rpm after which glutaraldehyde (1%) was slowly added for crosslinking of PTX molecules. The reaction mixture was kept under overnight stirring at 4 °C. After crosslinking process, the reaction mixture was centrifuged
PTX nano-assemblies structure was dispersed in PBS and seeded on glass slide in thin layer. The samples were allowed to air dry for few minutes before mounting with 90% glycerol in PBS. The fluorescence property of the molecule was imaged in all three channels (blue, green, 2
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Fig. 1. General procedure of PTX astral structure synthesis from PTX solution and fluorescence generation after glutaraldehyde mediated crosslinking. X1, X2 and X3 represent different structural parts of paclitaxel molecule.
and shown that PTX molecules self assembles into long ribbon-like or hollow fibrous structures in presence of water and organic solvent [24]. It was discovered that various factors could affect the self-assembly process of PTX astral structure, among which PTX concentration and solvent to antisolvent ratio are very crucial. Morphology of the PTX astral assemblies changes, with the change in concentration of PTX. This observation is in complete agreement with the earlier reports where similar morphology is reported in the presence of copolymers [23]. The currently reported hierarchical structure of PTX could only be achieved at a concentration of 2 mg/ml of PTX. The alteration in antisolvent: solvent ratio resulted in various morphological structures and sizes (Fig. 3a and b). The decrease in the solvent: antisolvent ratio from 3:7 to 3:9 resulted in the overall size reduction of the molecule with reduced central stalk and increase in the number of fibres. In the supernatant, no significant appearance of PTX nanoparticles was observed, clearly predicting the complete conversion of the pure PTX drug into the astral structure. The PTX molecules are known to interact among each other, and with the microenvironment of solvent ions present around the molecules, through an extensive network of hydrogen bonding [25]. To further validate the process NMR studies were carried out which presented significant changes in the chemical shifts of PTX when compared to self-assembled reaction intermediates (P2,P3) and PTX astral structure (P4). Alteration in chemical shift values could be of significant importance in the detection of the proton atoms that are contributing information of self-assembly of the molecule during synthesis. Fig. 4 shows the 1H NMR spectra of PTX in D6-DMSO at 298K for P1, P2, P3 and P4 respectively. Significant changes in the chemical shift values were observed at 4.69 ppm 6.16 ppm and 8.89 ppm assigned to the proton involvement of OH1, OH2'and 3′NH moieties. The proton peaks at 4.69 ppm and 6.16 ppm of OH1 and OH2′ moieties were found missing in case of the reaction intermediates (P2, P3) and final product of PTX self-assembled structure (P4). It could be speculated that the OH of C2′ and C1 position of P1 might be involved in cross-linking due to the presence of glutaraldehyde molecule. Appearance of additional
red). Seven day old HepG2 spheroids were seeded in 24-well plates and incubated with PTX astral assemblies for 6 h. After treatment, spheroids were washed three times with chilled PBS and fixed using 4% formaldehyde for 30 min at 4 °C. Multiple washing was performed after fixation to remove extra fixative. Spheroids were mounted on glass slides using 90% glycerol and coverslip. Control spheroids were stained with DAPI nuclear stain before mounting to glass slides. 3.8. Statistical analysis Statistical analyses were accomplished using the Origin 8.5 software. The data were statistically analysed using a one-way analysis of variance (ANOVA) at p < 0.01 level of significance. 4. Result and discussion PTX molecular structure could be divided into three parts (Fig. 1) among which X1 part of the molecule constitutes the core of the fibre structure, as a result of high repulsive forces acting between X2 and X3. Surprisingly no further report on the self-assembly of these PTX fibres is available. The formation of self-assembled structures of PTX could be due to the presence of very distinctive stereochemistry of the PTX molecule. The key feature lies in the structure of PTX itself due to its impressive molecular arrangement. The general procedure of supramolecular synthesis from the self-assembly of PTX molecules through antisolvent precipitation method is well described in Fig. 1. Scanning electron microscopy and confocal microscopy revealed the morphology of air-dried samples of self-assembled PTX structures which form long astral like assembly and multichannel fluorescence emission respectively. The dimensions of the astral structure were recorded to be ∼20 μm in diameter and ∼40 μm in length, while the individual fibre of the astral was ∼200 nm in diameter. Ethanolic solution of PTX gives a circular morphology (Fig. 2) with approximately 200 nm in diameter. The orientation of the PTX molecule is well projected and established in presence of water and organic solvent. It was computationally predicted 3
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Fig. 2. SEM image of air-dried paclitaxel solution (scale bar:2 μm, ×8000) (a), PTX astral structure (scale bar:20 μm, x700) (b), filaments of PTX astral structure (scale bar: 10 μm, x2500) (c), bright field image of PTX astral structure (scale bar:80 μm) (d) fluorescence confocal images of PTX astral structure in blue channel (e) green channel (f) red channel (g) merged image (scale bar: 40 μm) (h). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
vulnerable to leaching process, thereby reducing the efficacy of the method. In the current synthesis method, no external fluorescent molecule is introduced to the structure which nullifies the leaching phenomenon. The fluorescent property is the inbuilt character of the molecule. X-ray diffraction (XRD) patterns of PTX astral structures and pure PTX were recorded to study the crystal state of PTX. As shown in Fig. 3c, pure PTX shows main peaks at 2θ value of 5.22, 12.58 and 13.93 respectively, which are in coherence to the peaks present in PTX astral structure. The 13.93 2θ peak of PTX astral structure is splitted into two, thereby indicating the hydrated form of the PTX crystal. No further significant changes were observed in the XRD pattern of both the structures. This depicts negligible difference in the crystalline arrangement of PTX thus confirming the molecular integrity of the PTX molecule during synthesis. FTIR studies of PTX astral structure and PTX pure drug (Fig. 3e–h) were conducted to further analyse the possible mechanism associated with the formation of such structures. IR spectra of PTX and PTX astral structure presented a strong peak at 1646 cm−1 which could be
peak at 8.310 ppm further validates our hypothesis. The downfield shift of 3′NH from 8.89 to 8.94 in P3 and 8.96 in case of P4 specifies structural alteration which could be associated with intermolecular hydrogen bonding [26]. This alteration in chemical shift values strongly presents the presence of intermolecular hydrogen bonding as significant factor contributing to the astral assembly formation of PTX. The current method of synthesis utilizes glutaraldehyde as a crosslinking agent which could provide unique fluorescence properties and simultaneously improving the structural stability of the molecule. In the current synthesis, fluorescence property of the self-assembled structure of PTX could be attributed to the electronic transitions such as π-π* transition of C=C bond and n-π* transition of partial double bond characteristic of C=N bond assoiated with amide bonding [27]. This fluorescence property of structure is lost in the absence of the glutaraldehyde which signifies the importance of the molecule in the fluorescence generation. PTX nanocrystal doped with fluorescent guest molecules has been used earlier for the delivery and imaging application [28]. External introduction of fluorescence guest molecule into the structures, through ionic and physical adsorption method makes it
Fig. 3. SEM image of PTX astral structure prepared in solvent: antisolvent ration (3:7) scale bar:10 μm, x1200(a) ratio (3:9), scale bar: 10 μm, ×1400 (b) powder XRay diffraction (PXRD) of PTX microstructure and PTX Drug(c) In vitro percent drug release profile of PTX Astral structure and PTX drug (d) FTIR analysis of PTX astral structure and PTX pure drug showing various region of spectra from 400 to 1000 cm−1 (e) 1000-2000 cm−1 (f) 2000-4000 cm−1 (g) deconvoluted peaks of 1620-1695 cm−1 marked as two arrows in figure f, showing N–H stretching at 1646 cm−1 and C=O stretching at 1669 cm−1(h). 4
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Fig. 4. 1H NMR spectra of paclitaxel (P1), reaction intermediates (P2, P3), astral structure (P4) in DMSO solvent (a) enlarged view from chemical shift 4.0 to 6.5 (b) enlarged view from chemical shift 8.1 to 9.0 (c).
Although, its use in local delivery therapies like TACE is not much explored. Currently, PTX is in Phase I/II clinical trial (NCT03812874) in patients with non-resectable hepatocellular carcinoma following TACE treatment. In the current study therapeutic efficacy of the PTX in form of astral structure was evaluated on 3D spheroids model of HepG2 cell. 3D spheroid culture could provide a better therapeutic reality of molecule in comparison to the 2D monolayer model. Fig. 5a and b shows the therapeutic response of PTX astral structure at the clinically significant value of 10 nM PTX. Spheroid volume started reducing significantly from the 3rd day of treatment which improved further on the 5th day. The significant reduction in spheroid volume when compared to the control, validates the therapeutic efficacy of the molecule in complex systems like solid tumors. SEM images of PTX astral structure treated spheroid (Fig. 5c and d) further confirms the presence of the PTX assembly inside the spheroid on the 5th day of treatment, which justify the structural advantage associated with the astral structure of PTX in enhancing the accumulation of molecule inside the tumor during treatment. Fluorescence based imaging abilities of the astral assemblies of PTX were further explored after treating the 3D spheroid cultures of HepG2 liver carcinoma cell line (Fig. 6). The nucleus of the control HepG2 spheroids was stained using DAPI, while there was no fluorescent dye used in PTX astral structure treated spheroids. Confocal z stacked
assigned to C=O stretching vibration of amide bonding (-CONH-) present in PTX. The presence of 1624 cm−1 and 1669 cm−1 peaks could be accounted to the N–H stretching and C=O stretching respectively in the PTX astral structure. Region from 2900 to 3000 cm−1 represents the sp3 hybridized C–H bonds, while region from 3000 to 3100 cm−1 predicts the presence of sp2 hybridized C–H bonds in the case of PTX and PTX astral structure respectively. PTX pure drug shows sharp peaks at 3300 cm−1 and 3500 cm−1 which could be due to the presence of N–H stretching and O–H stretching respectively. The PTX astral structure also presents a broad peak of hydrogen-bonded O–H at 3333 cm−1 and free O–H at 3500 cm−1 respectively. This interaction indicated the remarkable role of hydrogen bonding in the formation of this unique assembly. The in-vitro release pattern of astral structure of PTX and pure PTX drug is presented in Fig. 3d. Sustained release pattern in case of PTX astral structure was observed when compared to pure PTX drug. This difference in the drug release profile could be attributed to the hydrated form of PTX which further supported our notion of sequestration of drug nanocrystal into one assembly. This could assist in slow release of the drug and further help in providing increased payload at the administration site with minimal dispersion to the surrounding. PTX has been successfully used independently [29] or in combination with other chemotherapeutic drugs in systemic deliveries to treat HCC [30]. 5
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Fig. 5. Time dependent brightfield images of Control (treated with PBS) and treatment (PTX astral assemblies) (a) quantification of effective volume change (b) SEM images of treated spheroid; scale bar:100 μm, ×120 (c) PTX astral structure inside spheroid; scale bar: 20 μm, ×950 (d).
images (Fig. S1) affirmed the in-depth distribution of fluorescence in spheroids. This could strengthen the significance of using PTX astral assemblies in TACE as it could allow anatomical fluorescence imaging in various surgical complications.
the tumor site and cent per cent loading makes it promising in its category of localized implantable chemotherapeutics. It could provide a structural advantage to the TACE procedures and can help in advancement against several disadvantage like MDR, hypoxia, and tumor recurrence.
5. Conclusion Funding sources In summary, a micron scale hierarchical PTX astral structure is synthesized through a self-assembly approach. Its unique morphology could be readily utilized for enhanced penetrance effect and localized application in solid tumors to overcome MDR and systemic toxicity related problems. The simple synthetic approach, fluorescence ability, higher therapeutic efficacy in the solid tumor, longer retention time at
This work is partially supported by the Science and Engineering Research Board (SERB) grants: YSS/2015/001706 and ECR/2016/ 000633/LS; Department of Biotechnology grants: 102/IFD/SAN/1734/ 2016–2017 and BT/PR27008/NNT/28/1525/-2017 and DST Nano Mission grant: SR/NM/NB-1108/2016 (G). The University Grant Fig. 6. Confocal images of Control HepG2 spheroids stained with DAPI in brightfield channel (a), DAPI channel (b), FITC channel (c), TRITC channel (d), PTX astral structure treated HepG2 spheroids without DAPI staining in bright field (e), DAPI channel (f), FITC channel (g), TRITC channel (h). All images showing scale bar of 200micron. (Note: The fluorescence in Treatment group (f,g,h) is due to the autofluorescence property of PTX astral assemblies penetrated inside the spheroids).
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Materials Science & Engineering C 107 (2020) 110315
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Commission JRF in Science Fellowship (Sr. No. 2121330542 Ref no. 22/12/2013 (ii) EU-V) to Atul Dev is generously acknowledged.
[10] X.Z. Wu, G.R. Xie, D. Chen, J. Gastroenterol. Hepatol. 22 (2007) 1178–1182. [11] M. Tezuka, K. Hayashi, K. Kubota, S. Sekine, Y. Okada, H. Ina, T. Irie, Dig. Dis. Sci. 52 (2007) 783–788. [12] J.P. Piret, J.P. Cosse, N. Ninane, M. Raes, C. Michiels, Exp. Cell Res. 312 (2006) 2908–2920. [13] M.A. Jordan, L. Wilson, Nat. Rev. Cancer 4 (2004) 253–265. [14] H. Gelderblom, J. Verweij, K. Nooter, A. Sparreboom, Eur. J. Cancer 37 (2001) 1590–1598. [15] Y. Lu, Z.H. Wang, T. Li, H. McNally, K. Park, M. Sturek, J. Control. Release : Off. J. Control. Release Soc. 176 (2014) 76–85. [16] K.A. Gawde, S. Sau, K. Tatiparti, S.K. Kashaw, M. Mehrmohammadi, A.S. Azmi, A.K. Iyer, Colloids Surfaces B Biointerfaces 167 (2018) 8–19. [17] H. Yu, Y. Wang, S. Wang, X. Li, W. Li, D. Ding, X. Gong, M. Keidar, W. Zhang, ACS Applied Materials & Interfaces, (2018). [18] P. Ma, R.J. Mumper, J. Nanomed. Nanotechnol. 4 (2013) 1000164. [19] R.H. Muller, S. Gohla, C.M. Keck, Eur. J. Pharm. Biopharm. : Off. J. Arbeitsgemeinschaft Fur Pharmazeutische Verfahrenstechnik E.V 78 (2011) 1–9. [20] Y. Liu, L. Huang, F. Liu, Mol. Pharm. 7 (2010) 863–869. [21] L. Wei, Y. Ji, W. Gong, Z. Kang, M. Meng, A. Zheng, X. Zhang, J. Sun, Drug Dev. Ind. Pharm. 41 (2015) 1343–1352. [22] S.E. Lee, S.F. Bairstow, J.O. Werling, M.V. Chaubal, L. Lin, M.A. Murphy, J.P. DiOrio, J. Gass, B. Rabinow, X. Wang, Y. Zhang, Z. Yang, R.M. Hoffman, Pharm. Dev. Technol. 19 (2014) 438–453. [23] J.P. Tan, S.H. Kim, F. Nederberg, E.A. Appel, R.M. Waymouth, Y. Zhang, J.L. Hedrick, Y.Y. Yang, Small 5 (2009) 1504–1507. [24] X.D. Guo, J.P. Tan, S.H. Kim, L.J. Zhang, Y. Zhang, J.L. Hedrick, Y.Y. Yang, Y. Qian, Biomaterials 30 (2009) 6556–6563. [25] D. Mastropaolo, A. Camerman, Y. Luo, G.D. Brayer, N. Camerman, Proc. Natl. Acad. Sci. U. S. A 92 (1995) 6920–6924. [26] S.V. Balasubramanian, J.L. Alderfer, R.M. Straubinger, J. Pharm. Sci. 83 (1994) 1470–1476. [27] X. Ma, X. Sun, D. Hargrove, J. Chen, D. Song, Q. Dong, X. Lu, T.H. Fan, Y. Fu, Y. Lei, Sci. Rep. 6 (2016) 19370. [28] R. Zhao, C.P. Hollis, H. Zhang, L. Sun, R.A. Gemeinhart, T. Li, Mol. Pharm. 8 (2011) 1985–1991. [29] L. Chen, Y. Liu, W. Wang, K. Liu, Oncol. Lett. 10 (2015) 77–84. [30] C. Jin, H. Li, Y. He, M. He, L. Bai, Y. Cao, W. Song, K. Dou, J. Cancer Res. Clin. Oncol. 136 (2010) 267–274.
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Paclitaxel nanocrystalline assemblies as a potential transcatheter arterial chemoembolization (TACE) candidate for unresectable hepatocellular carcinoma
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Atul Dev, Ankur Sood, Subhasree Roy Choudhury, Surajit Karmakar∗ Institute of Nano Science and Technology, Mohali, Punjab, India
A B S T R A C T
We have prepared a water dispersible Nanocrystalline hierarchical structure of paclitaxel using self-assembly approach. Its unique structure provides the advantage of increased payload and higher retention of a chemotherapeutic drug inside the tumor site for a prolonged period. Micron scale structure, sustained release behaviour, multichannel fluorescence property and devoid of any vehicle molecule makes it promising in treating solid tumors through localized drug delivery approach like TACE. This method is based on one pot synthesis through co-precipitation reaction followed by crosslinking. Therapeutic efficacy of the molecules is successfully verified through the significant reduction in the volume of the 3D spheroid model of hepatocellular carcinoma (HCC).
1. Introduction Hepatocellular carcinoma (HCC) is the primary form of Liver cancer that originates in the liver. It is the third leading cause of cancer-related deaths and represents 70–85% cases of primary liver cancers worldwide [1]. Despite therapeutic advances, liver cancer remains one of the most challenging forms of cancers to treat. Surgical resection is the primary treatment for BCLC (Barcelona clinic liver cancer) stage A patients characterized by solitary lesion > 2 cm or up to 3 multifocal lesions measuring less than 3 cm. Sorafenib, a multiple kinase inhibitor is the first line therapy to treat BCLC stage C patients who have reduced hepatic functions with vascular invasion or distant metastasis. Patients, who fail Sorafenib treatment has shown significant improvement in survival using Regorafenib [2]. Although, development of second line systemic therapies after Sorafenib is still in early stages. Lack of selectivity, recurrence, multidrug resistance and severe side effects of systemic chemotherapy significantly limits the success rate in the treatment of HCC. Therefore, it is of great clinical importance to develop new strategies to overcome these issues. Local drug delivery approaches could provide benefits of minimal systemic toxicity, an increased payload of the drug with attenuation of multidrug resistance (MDR) [3]. Of lately, Transcatheter arterial chemoembolization (TACE) has become the most common approach for the management of HCC in BCLC stage B patients. It is often recommended for BCLC stage C patients along with Sorafenib treatment to further improve the survival. It combines the injection of chemotherapeutic drug with simultaneous
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occlusion of the tumor blood supply. HCC exhibits intense neoangiogenic activity and is mostly dependent on the hepatic artery for blood supply, while the rest of the liver is supplied by the portal vein [4]. This provides the rationale to use arterial obstruction as an effective therapeutic option, as it induces ischemic tumor necrosis. TACE showed increased survival rate in comparison to other best supportive care [5–9]. Despite being the effective treatment method, TACE suffers from some serious drawbacks such as generation of hypoxia condition during embolization which in turn leads to aggressive phenotypes of tumor with increased angiogenesis and metastasis activity [10]. The chemoresistance and recurrence of HCC are other critical problems associated with tumor hypoxia [11,12]. As a result of this, complete neutralization of tumor cells is not possible by chemotherapeutic drugs being used in TACE. To achieve significant survival in TACE, improvement in current approaches is immensely required. Effective killing along with negligible generation of hypoxia condition is the prerequisite for a formulation to be notably effective for its utilization in TACE. Currently, the most common anticancer drug used in TACE is doxorubicin (36%), followed by cisplatin (31%), epirubicin/doxorubicin (12%), mitoxantrone (8%), mitomycin C (8%) and a chemical conjugate of a synthetic copolymer of styrene maleic acid (SMA) and the proteinaceous anti-cancer agent neocarzinostatin (NCS) SMANCS (5%) [8]. Interestingly, Paclitaxel (PTX) which is one of the most effective drugs against lung, ovarian, and breast cancer [13] has not been frequently used in TACE owing to its current commercial formulation available which includes organic solvents polyoxyethylated castor oil
Corresponding author. Institute of Nano Science and Technology, Habitat Centre, Phase 10, Sector 64, Sahibzada Ajit Singh Nagar, Punjab, India. E-mail address: [email protected] (S. Karmakar).
https://doi.org/10.1016/j.msec.2019.110315 Received 30 January 2019; Received in revised form 1 October 2019; Accepted 14 October 2019 Available online 17 October 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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at 10000 g for 30min and was washed three times using double distilled water and lyophilized for further use.
(Cremophor EL) and dehydrated ethanol (1:1, v/v). Theses organic contents are reported to induce severe toxic effects which include neurotoxicity, hypersensitivity, cardio-toxicity, anaphylaxis and nephrotoxicity [14]. Thus various alternative nano platforms like drug nanocrystals [15], polymers [16], lipids, drug polymer conjugates, magnetic nanoparticles [17], inorganic nanoparticles etc. were investigated for the delivery of PTX in different cancer models including HCC. Despite improved attributes of nano-delivery systems, systemic clearance and non-specific distribution in organs still impede the clinical usage of nano-formulations in TACE [18]. This work reports the synthesis of a novel structural assembly of paclitaxel nanocrystals, which could be directly introduced into the tumor site with the help of a catheter. Drug nanocrystals gained interest in delivering poorly water-soluble anticancer drugs due to the high drug loading efficiency, circulation stability [19] and ability to resolve multidrug resistance [20]. The prominent advantage of nanocrystals is the absence of delivery vehicle that may introduce adverse or unwanted toxic reactions into the human body. The current approach well suits for PTX to produce Cremophor EL®-free formulations. Several studies on PTX nanocrystals have been carried out for the treatment of various cancer [21,22] which signify its therapeutic efficacy. The unique structural arrangements of PTX nanocrystals could permit improved chemotherapeutic effect through increased payload of the drug and simultaneous occlusion of the blood flow with minimum hypoxia effect. This structural assembly could find its utilization in TACE therapy as a potential chemotherapeutic agent. The current work addresses the challenges of systemic delivery and TACE procedures. We have reported a highly fluorescent, water dispersible, nanocrystalline assemblies of PTX drug which shows sustained drug release profile as compared to the bare PTX molecule. Inbuilt multichannel fluorescent properties make these particles expedient during surgical procedures, the ability of real-time monitoring of such particles in the course of catheter mediated delivery could provide edge over currently available formulations. These PTX astral assemblies could provide structural advantage over currently available TACE formulations. Its unique astral appearance promises prolonged retention inside tumor with minimum passive diffusion to the nearby healthy tissues. Current hierarchical self-assembled PTX structures closely resemble with earlier reported structures [23] but are unique in its ease of one pot synthesis, morphology and inherent fluorescence property. One pot synthesis of such structures allows structural uniformity and ease of scaleup during large scale production.
3.2. Characterization X-ray diffractograms (XRD) were recorded on a Bruker powder XRD D8 X-ray diffractometer over 2θ range of 5°–40° using Cu Kα X-rays of wavelength1.54 Å operated at 30 mA & 40 kV. Scanning electron microscopy (SEM) images were obtained with JEOL at 10–20 kV. Fourier transform infrared (FTIR) spectra were recorded on Cary Agilent 660 IR spectrophotometer by mixing the samples with potassium bromide and making pellets for analysis. For each spectrum, 256 scans and 4 cm−1 resolution were applied over the range of 400–4000 cm−1. UV-Visible spectrophotometer Shimadzu UV-2600 was used to estimate the drug release. Fluorescence images were captured using Zeiss confocal laser scanning microscope. 3.3. 1H NMR studies Proton (1H)-NMR studies were carried out for the analysis of anhydrous PTX (P1), reaction intermediates (P2 and P3) and PTX astral assemblies (P4) respectively. Samples to be investigated were dissolved in D-6 DMSO and analysed using Bruker Av III HD at 400 MHz and 25 °C. The chemical shift values are reported in ppm scale using tetramethylsilane (TMS) as an internal standard. 3.4. In-vitro drug release studies In vitro drug release studies were performed in PBS pH 7.4 using 12KD cut-off dialysis membrane. One mg of PTX and PTX nano-assemblies were weighed separately and suspended in 1 ml of PBS solution containing 0.1% of SLS. Drug release was analysed in 20 ml of PBS solution as a releasing media at 37 °C with stirring at 150 rpm. At different time intervals one ml of aliquot was taken, and sink was replaced with the same amount of solvent, aliquot was analysed using UV–Vis spectrophotometer after preparing standard curve of PTX in methanol at 231 nm. 3.5. Spheroid generation and treatment Spheroid was generated using a hanging drop culture technique. HepG2 cells were cultured in DMEM media supplemented with 10% FBS maintaining 2000 cells per hanging drop. After 7 days of culture at 37 °C and 5% CO2, spheroids were collected for treatment. Spheroids were then treated with a clinically relevant concentration of PTX (10 nM) in form of astral structures and imaged at various time points. Scanning electron microscopy was performed to verify the presence of astral structure inside the spheroid.
2. Materials and methods 2.1. Materials Paclitaxel (PTX) was purchased from Clear synth. Sodium lauryl sulfate (SLS), Ethanol molecular grade was purchased from Merck. Glutaraldehyde 25% w/v solution, Dulbecco's Modified Eagle Medium (DMEM), streptomycin penicillin solution, Fetal Bovine Serum (FBS) and Trypsin EDTA were procured from HI-media. DAPI(4′,6-diamidino2-phenylindole), Glycerol, and 12KD dialysis membrane were purchased from sigma Aldrich. Human liver carcinoma cell line (HepG2) was purchased from National Centre for Cell Science, Pune.
3.6. Scanning electron microscopy of spheroids Hep G2 spheroids after 5-day treatment with PTX nano-assemblies were imaged using Scanning electron microscopy. The treated spheroids were washed 3–4 times with chilled PBS and fixed overnight in paraformaldehyde at 4 °C. After overnight fixation the spheroids were washed three times with chilled PBS and subsequently treated with gradient series of ethanol. The spheroids were broken to expose the internal morphology and presence of PTX astral assemblies. Finally, they were sputter-coated with gold and imaged at 10 kV.
3. Methods 3.1. Synthesis of PTX astral structure
3.7. Confocal laser fluorescence microscopy
Paclitaxel was dissolved in ethanol (2 mg/ml) and added dropwise to the antisolvent (water) while maintaining the solvent and antisolvent ratio (3:7). The system was stirred continuously for 15 min at 1000 rpm after which glutaraldehyde (1%) was slowly added for crosslinking of PTX molecules. The reaction mixture was kept under overnight stirring at 4 °C. After crosslinking process, the reaction mixture was centrifuged
PTX nano-assemblies structure was dispersed in PBS and seeded on glass slide in thin layer. The samples were allowed to air dry for few minutes before mounting with 90% glycerol in PBS. The fluorescence property of the molecule was imaged in all three channels (blue, green, 2
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Fig. 1. General procedure of PTX astral structure synthesis from PTX solution and fluorescence generation after glutaraldehyde mediated crosslinking. X1, X2 and X3 represent different structural parts of paclitaxel molecule.
and shown that PTX molecules self assembles into long ribbon-like or hollow fibrous structures in presence of water and organic solvent [24]. It was discovered that various factors could affect the self-assembly process of PTX astral structure, among which PTX concentration and solvent to antisolvent ratio are very crucial. Morphology of the PTX astral assemblies changes, with the change in concentration of PTX. This observation is in complete agreement with the earlier reports where similar morphology is reported in the presence of copolymers [23]. The currently reported hierarchical structure of PTX could only be achieved at a concentration of 2 mg/ml of PTX. The alteration in antisolvent: solvent ratio resulted in various morphological structures and sizes (Fig. 3a and b). The decrease in the solvent: antisolvent ratio from 3:7 to 3:9 resulted in the overall size reduction of the molecule with reduced central stalk and increase in the number of fibres. In the supernatant, no significant appearance of PTX nanoparticles was observed, clearly predicting the complete conversion of the pure PTX drug into the astral structure. The PTX molecules are known to interact among each other, and with the microenvironment of solvent ions present around the molecules, through an extensive network of hydrogen bonding [25]. To further validate the process NMR studies were carried out which presented significant changes in the chemical shifts of PTX when compared to self-assembled reaction intermediates (P2,P3) and PTX astral structure (P4). Alteration in chemical shift values could be of significant importance in the detection of the proton atoms that are contributing information of self-assembly of the molecule during synthesis. Fig. 4 shows the 1H NMR spectra of PTX in D6-DMSO at 298K for P1, P2, P3 and P4 respectively. Significant changes in the chemical shift values were observed at 4.69 ppm 6.16 ppm and 8.89 ppm assigned to the proton involvement of OH1, OH2'and 3′NH moieties. The proton peaks at 4.69 ppm and 6.16 ppm of OH1 and OH2′ moieties were found missing in case of the reaction intermediates (P2, P3) and final product of PTX self-assembled structure (P4). It could be speculated that the OH of C2′ and C1 position of P1 might be involved in cross-linking due to the presence of glutaraldehyde molecule. Appearance of additional
red). Seven day old HepG2 spheroids were seeded in 24-well plates and incubated with PTX astral assemblies for 6 h. After treatment, spheroids were washed three times with chilled PBS and fixed using 4% formaldehyde for 30 min at 4 °C. Multiple washing was performed after fixation to remove extra fixative. Spheroids were mounted on glass slides using 90% glycerol and coverslip. Control spheroids were stained with DAPI nuclear stain before mounting to glass slides. 3.8. Statistical analysis Statistical analyses were accomplished using the Origin 8.5 software. The data were statistically analysed using a one-way analysis of variance (ANOVA) at p < 0.01 level of significance. 4. Result and discussion PTX molecular structure could be divided into three parts (Fig. 1) among which X1 part of the molecule constitutes the core of the fibre structure, as a result of high repulsive forces acting between X2 and X3. Surprisingly no further report on the self-assembly of these PTX fibres is available. The formation of self-assembled structures of PTX could be due to the presence of very distinctive stereochemistry of the PTX molecule. The key feature lies in the structure of PTX itself due to its impressive molecular arrangement. The general procedure of supramolecular synthesis from the self-assembly of PTX molecules through antisolvent precipitation method is well described in Fig. 1. Scanning electron microscopy and confocal microscopy revealed the morphology of air-dried samples of self-assembled PTX structures which form long astral like assembly and multichannel fluorescence emission respectively. The dimensions of the astral structure were recorded to be ∼20 μm in diameter and ∼40 μm in length, while the individual fibre of the astral was ∼200 nm in diameter. Ethanolic solution of PTX gives a circular morphology (Fig. 2) with approximately 200 nm in diameter. The orientation of the PTX molecule is well projected and established in presence of water and organic solvent. It was computationally predicted 3
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Fig. 2. SEM image of air-dried paclitaxel solution (scale bar:2 μm, ×8000) (a), PTX astral structure (scale bar:20 μm, x700) (b), filaments of PTX astral structure (scale bar: 10 μm, x2500) (c), bright field image of PTX astral structure (scale bar:80 μm) (d) fluorescence confocal images of PTX astral structure in blue channel (e) green channel (f) red channel (g) merged image (scale bar: 40 μm) (h). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
vulnerable to leaching process, thereby reducing the efficacy of the method. In the current synthesis method, no external fluorescent molecule is introduced to the structure which nullifies the leaching phenomenon. The fluorescent property is the inbuilt character of the molecule. X-ray diffraction (XRD) patterns of PTX astral structures and pure PTX were recorded to study the crystal state of PTX. As shown in Fig. 3c, pure PTX shows main peaks at 2θ value of 5.22, 12.58 and 13.93 respectively, which are in coherence to the peaks present in PTX astral structure. The 13.93 2θ peak of PTX astral structure is splitted into two, thereby indicating the hydrated form of the PTX crystal. No further significant changes were observed in the XRD pattern of both the structures. This depicts negligible difference in the crystalline arrangement of PTX thus confirming the molecular integrity of the PTX molecule during synthesis. FTIR studies of PTX astral structure and PTX pure drug (Fig. 3e–h) were conducted to further analyse the possible mechanism associated with the formation of such structures. IR spectra of PTX and PTX astral structure presented a strong peak at 1646 cm−1 which could be
peak at 8.310 ppm further validates our hypothesis. The downfield shift of 3′NH from 8.89 to 8.94 in P3 and 8.96 in case of P4 specifies structural alteration which could be associated with intermolecular hydrogen bonding [26]. This alteration in chemical shift values strongly presents the presence of intermolecular hydrogen bonding as significant factor contributing to the astral assembly formation of PTX. The current method of synthesis utilizes glutaraldehyde as a crosslinking agent which could provide unique fluorescence properties and simultaneously improving the structural stability of the molecule. In the current synthesis, fluorescence property of the self-assembled structure of PTX could be attributed to the electronic transitions such as π-π* transition of C=C bond and n-π* transition of partial double bond characteristic of C=N bond assoiated with amide bonding [27]. This fluorescence property of structure is lost in the absence of the glutaraldehyde which signifies the importance of the molecule in the fluorescence generation. PTX nanocrystal doped with fluorescent guest molecules has been used earlier for the delivery and imaging application [28]. External introduction of fluorescence guest molecule into the structures, through ionic and physical adsorption method makes it
Fig. 3. SEM image of PTX astral structure prepared in solvent: antisolvent ration (3:7) scale bar:10 μm, x1200(a) ratio (3:9), scale bar: 10 μm, ×1400 (b) powder XRay diffraction (PXRD) of PTX microstructure and PTX Drug(c) In vitro percent drug release profile of PTX Astral structure and PTX drug (d) FTIR analysis of PTX astral structure and PTX pure drug showing various region of spectra from 400 to 1000 cm−1 (e) 1000-2000 cm−1 (f) 2000-4000 cm−1 (g) deconvoluted peaks of 1620-1695 cm−1 marked as two arrows in figure f, showing N–H stretching at 1646 cm−1 and C=O stretching at 1669 cm−1(h). 4
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Fig. 4. 1H NMR spectra of paclitaxel (P1), reaction intermediates (P2, P3), astral structure (P4) in DMSO solvent (a) enlarged view from chemical shift 4.0 to 6.5 (b) enlarged view from chemical shift 8.1 to 9.0 (c).
Although, its use in local delivery therapies like TACE is not much explored. Currently, PTX is in Phase I/II clinical trial (NCT03812874) in patients with non-resectable hepatocellular carcinoma following TACE treatment. In the current study therapeutic efficacy of the PTX in form of astral structure was evaluated on 3D spheroids model of HepG2 cell. 3D spheroid culture could provide a better therapeutic reality of molecule in comparison to the 2D monolayer model. Fig. 5a and b shows the therapeutic response of PTX astral structure at the clinically significant value of 10 nM PTX. Spheroid volume started reducing significantly from the 3rd day of treatment which improved further on the 5th day. The significant reduction in spheroid volume when compared to the control, validates the therapeutic efficacy of the molecule in complex systems like solid tumors. SEM images of PTX astral structure treated spheroid (Fig. 5c and d) further confirms the presence of the PTX assembly inside the spheroid on the 5th day of treatment, which justify the structural advantage associated with the astral structure of PTX in enhancing the accumulation of molecule inside the tumor during treatment. Fluorescence based imaging abilities of the astral assemblies of PTX were further explored after treating the 3D spheroid cultures of HepG2 liver carcinoma cell line (Fig. 6). The nucleus of the control HepG2 spheroids was stained using DAPI, while there was no fluorescent dye used in PTX astral structure treated spheroids. Confocal z stacked
assigned to C=O stretching vibration of amide bonding (-CONH-) present in PTX. The presence of 1624 cm−1 and 1669 cm−1 peaks could be accounted to the N–H stretching and C=O stretching respectively in the PTX astral structure. Region from 2900 to 3000 cm−1 represents the sp3 hybridized C–H bonds, while region from 3000 to 3100 cm−1 predicts the presence of sp2 hybridized C–H bonds in the case of PTX and PTX astral structure respectively. PTX pure drug shows sharp peaks at 3300 cm−1 and 3500 cm−1 which could be due to the presence of N–H stretching and O–H stretching respectively. The PTX astral structure also presents a broad peak of hydrogen-bonded O–H at 3333 cm−1 and free O–H at 3500 cm−1 respectively. This interaction indicated the remarkable role of hydrogen bonding in the formation of this unique assembly. The in-vitro release pattern of astral structure of PTX and pure PTX drug is presented in Fig. 3d. Sustained release pattern in case of PTX astral structure was observed when compared to pure PTX drug. This difference in the drug release profile could be attributed to the hydrated form of PTX which further supported our notion of sequestration of drug nanocrystal into one assembly. This could assist in slow release of the drug and further help in providing increased payload at the administration site with minimal dispersion to the surrounding. PTX has been successfully used independently [29] or in combination with other chemotherapeutic drugs in systemic deliveries to treat HCC [30]. 5
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Fig. 5. Time dependent brightfield images of Control (treated with PBS) and treatment (PTX astral assemblies) (a) quantification of effective volume change (b) SEM images of treated spheroid; scale bar:100 μm, ×120 (c) PTX astral structure inside spheroid; scale bar: 20 μm, ×950 (d).
images (Fig. S1) affirmed the in-depth distribution of fluorescence in spheroids. This could strengthen the significance of using PTX astral assemblies in TACE as it could allow anatomical fluorescence imaging in various surgical complications.
the tumor site and cent per cent loading makes it promising in its category of localized implantable chemotherapeutics. It could provide a structural advantage to the TACE procedures and can help in advancement against several disadvantage like MDR, hypoxia, and tumor recurrence.
5. Conclusion Funding sources In summary, a micron scale hierarchical PTX astral structure is synthesized through a self-assembly approach. Its unique morphology could be readily utilized for enhanced penetrance effect and localized application in solid tumors to overcome MDR and systemic toxicity related problems. The simple synthetic approach, fluorescence ability, higher therapeutic efficacy in the solid tumor, longer retention time at
This work is partially supported by the Science and Engineering Research Board (SERB) grants: YSS/2015/001706 and ECR/2016/ 000633/LS; Department of Biotechnology grants: 102/IFD/SAN/1734/ 2016–2017 and BT/PR27008/NNT/28/1525/-2017 and DST Nano Mission grant: SR/NM/NB-1108/2016 (G). The University Grant Fig. 6. Confocal images of Control HepG2 spheroids stained with DAPI in brightfield channel (a), DAPI channel (b), FITC channel (c), TRITC channel (d), PTX astral structure treated HepG2 spheroids without DAPI staining in bright field (e), DAPI channel (f), FITC channel (g), TRITC channel (h). All images showing scale bar of 200micron. (Note: The fluorescence in Treatment group (f,g,h) is due to the autofluorescence property of PTX astral assemblies penetrated inside the spheroids).
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Commission JRF in Science Fellowship (Sr. No. 2121330542 Ref no. 22/12/2013 (ii) EU-V) to Atul Dev is generously acknowledged.
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