In vitro evaluation and characterization of cisplatin loaded nano�bers for local chemotherapy

Cancer is a disease that affects the quality of life of the patients that are treated with Cisplatin (CDDP), which is needed for adjuvant therapy, however it leads to many secondary and adverse effects. In this study, we manufactured and characterized poly-(lactic acid) (PLA) non-woven �bers charged with Cisplatin (CDDP) by electrospinning technique to evaluate their cytotoxicity in in vitro assays on HeLa cells (Cervical Carcinoma Cell Line). PLA – CDDP solutions with increasing concentrations of CDDP (0.5, 1 and 2 % w/w) were used in a TL-01 electrospinning equipment with the same system parameters. We analyzed the chemical, thermal and morphological characteristics of PLA and PLA – CDDP �ber mats. Furthermore, hydrolytic degradation, haemolysis and toxicity in HeLa cells were evaluated. By adding the CDDP to the �bers, the degradation, glass transition and melting temperatures were modi�ed; the 3 µm �ber diameter of pristine PLA �bers was decreased in half the size and the degradation time was extended over 5 months. However, the hemocompatibility of the material with and without CDDP was mantained, while cytotoxicity in HeLa cells increased in the three concentrations of �ber mats of PLA – CDDP compared to the intravenous drug at 24 h (P = ). We concluded that the �ber mats PLA – CDDP could be used for localized treatment in the adjuvant treatment when resection panels are expose after a surgical extirpation of solid tumors


Introduction
Cancer is a disease caused by uncontrolled cell growth and division in a tissue, and it can appear anywhere in the body [1].It is the sixth leading cause of death around the world with 18 million new cases only in 2018.In Mexico in the same year, the cases increased to 190,600, with a projected increase in the next 20 years [2][3][4].
Chemotherapy is part of the treatment regimen to combat this disease in order to shrink the tumor or eliminate metastatic cells in the system (neoadjuvant or adjuvant therapy) [5][6][7].Cisplatin (CDDP), one of the drugs used in these therapies, is characterized by being a chelating antineoplastic agent, recommended as an adjuvant in metastatic lung, liver or stomach cancer, although it is also used for cervical and head-neck cancer [ 8.9].
Cisplatin exerts its activity upon entering the cell, and its mechanism of action is based on acting as an electrophilic molecule, being able to bind to thiol groups of enzymes and structural proteins in addition to forming adducts in the DNA, thus causing cellular death [10].It is worth mentioning that because this is applied intravenously (IV), its distribution is throughout the body, causing adverse effects such as nausea, vomiting, nephrotoxicity, anemia, cardiotoxicity and long-term sequelae in the patient's life [11][12][13][14][15][16], in addition to the fact that cells can generate resistance to these drugs [17][18][19].That is why, one of the challenges of chemotherapy is to reduce systemic toxicity, and one way to approach this end is through the use of Drug Delivery Systems (DDS).
For example, Zong et al. [20] obtained bers from PLA and poly (ethylene oxide) (PEO) by electrospinning, encapsulating CDDP within the bers, which was then tested in a murine model.The results were evaluated by the reducing weight of the tumor in the cervix, likewise, the organs were extracted for the quanti cation of CDDP.The in situ treatments with bers charged with CDDP and the drug administered by IV route, at the same doses, were compared.The tumors weighed the same after the treatments, with the difference that the therapy with the charged bers decreased the amount of CDDP in organs such as kidneys, heart and liver, and high concentrations were found in the tumor and peritumoral area.The opposite happened with the IV treatment, demonstrating the safety of the bers in situ.
DDS have also shown that due to their immediate and recurrent action in the place where they should exert their activity, they can prevent cells from acquiring resistance to antineoplastic drugs, avoiding therapeutic failure [21][22][23].
There are several ways to design a ber-based DDS, which can be through template-assisted synthesis techniques, self-assembly, solvent casting, phase separation and electrospinning, the latter being the most widely used for providing greater surface area due to pores formed in the bers and for ease of use.
In the electrospinning process, polymeric solutions are used, made from synthetic polymers, biopolymers or copolymers, for the creation of bers conjugated to drugs, genes, nanoparticles, excitatory molecules by physical means, among others.[24][25][26][27][28].This method is in uenced by the process conditions, the concentrations and characteristics of the polymers used [29,30].
Biodegradable polymers are currently used in these processes due to their degradation and their compatibility with the human body [31][32][33].Poly (lactic acid) (PLA) is one of the most widely used materials, for example, in implants, orthopedic equipment and tissue engineering, because it degrades by hydrolysis or enzymes, which then, the L-lactic acid monomers are absorbed by cells and used in the Krebs cycle until they are eliminated as CO 2 and H 2 O. Furthermore, it has been observed in medical devices, based on PLA, that it does not generate immunogenicity when inside the body [34][35][36][37].
Thus, the objective of the present work is to manufacture poly (lactic acid) (PLA) bers charged with different concentrations of Cisplatin (CDDP) by electrospinning technique, to characterize their morphological, chemical, and thermal properties and to evaluate their cytotoxicity in in vitro tests with HeLa cervical cancer cells.

Materials
Poly (L-lactic acid) (PLA) in 3D printing lament and Cisplatin (CDDP) obtained from Sigma Aldrich, chloroform (JT BAKER) and dimethylformamide (Biopack) were used.A cervical cancer cell line, HeLa ATCC CCL-2, was used for cytotoxic evaluation.

System parameters
The PLA solution consisted of a mixture of dimethylformamide (DMF) and chloroform (CCl 3 ) in a ratio of 2:8, and the polymer in a concentration of 8% w/w.For the PLA -CDDP solutions, the different doses of CDDP were weighted in an analytical balance, corresponding to 0.5 (8.28 mg), 1 (16.56 mg) and 2 (22.09mg) % of the weight of PLA used.The CDDP was dissolved in de DMF volume for 6 h of constant stirring.
Then, the PLA solution was added and let for stirring overnight at room temperature for homogeneity purposes.

Electrospinning process
The polymers solutions (P0Pt, P1Pt, P2Pt and P3Pt) were added to a 20 mL syringe, which was then used in the TL-01 electrospinning apparatus from Tong Li Tech.The conditions used were modi ed from those obtained by Gutiérrez et al. [38] due to the addition of DMF and CDDP.A volume of 6 mL was used; ow rate of 3 mL / h; rotating collector plate moving speed of 200 rpm; axial speed of 2 mm / s; 7-Gauge needle and the distance between the tip of the needle and the collector roll was 15 cm.

Characterizaton
A Thermo Scienti c Nicolet 1510 ATR-Diamond kit was used to obtain the infrared spectra of ber mat samples.The spectra were obtained by ATR mode using a resolution of 4 with 64 scans in the spectral range between 400-4000 cm − 1 .For thermal characterization, Differential Scanning Calorimetry (DSC) was used with a DSC Q2000 equipment from TA Instruments Inc., in which 10 mg of sample was encapsulated and subjected to two heating cycles at 5 ° C / min from − 10 to 180 ° C under N 2 atmosphere.Likewise, Thermogravimetric Analysis (TGA) was performed using a Perkin Elmer TGA 8000 equipment.In this process, a 7 mg sample was subjected to a heating rate from 50 to 650 ° C with a rate of 10 ° C / min under an atmosphere of N 2 .

Morphology
Electrospun bers were observed using Scanning Electron Microscopy (SEM) in a Helios G4CX equipment at a voltage of 30 kV.At least 10 different micrographs of the sample were taken.Previously, the meshes were coated with gold by means of cathodic assisted sputtering.The micrographs were used to analyze the distribution of the ber diameters and the average of these was analyzed using ImageJ software (National Institutes of Health, USA).

Degradation test
Samples of 1 cm 2 were weighed and placed in conical tubes with 5 mL of sterile Phosphate Buffer Saline (PBS) (pH 7.44) and left in constant stirring at 37 ° C for a period of 0, 1, 3 and 5 months, with PBS change every week.The pH of the medium and weight loss was measured [39].

Haemolysis test
A 1:50 solution of erythrocytes and sterile PBS (pH 7.2) was obtained.Then, 1 cm 2 samples of the bers were prepared and allowed to soak for 30 min in PBS, prior to contact with erythrocytes.Subsequently, they were added to the solution for 1 h with constant stirring.They were then centrifuged at 5,000 x g for 5 min and the absorbance at 541 nm was read.The percentage of hemolysis obtained with the following equation was reported: Where OD is the absorbance value of the positive and negative controls and of the sample.

Assays in cell culture
The HeLa cervical cancer cell line was used.The cells were cultured in DMEM medium, supplemented with 10% inactivated fetal bovine serum (FBS) and 1% penicillin / gentamicin (AB).They were incubated at 37 ° C in an atmosphere of 95% air, 5% CO 2 and 98% humidity.

Cell viability study
HeLa cells were cultured in a 96-well microculture plate, at a concentration of 5 x 10 5 cells / well in DMEM / SFB / AB medium, incubated for 24 h at 37 ± 0.5 ° C. Subsequently, a disk of approximately 1 mg of bers charged with and without CDDP was added to the wells with medium and HeLa cells, which were incubated for 24 h.The percent growth inhibition was measured using MTT [3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltratazolium bromide].

Statistic analysis
Data are expressed as arithmetic mean with standard deviation.Statistical analysis was performed using ANOVA analysis with Dunett's multiple comparison post hoc test, the level of signi cance was de ned as p < 0.05.The analyzes were performed using the GraphPad Prism program (version 6 for Microsoft Windows).

Results
We obtained 4 ber mats with different concentrations of CDDP, the P0Pt, P1Pt, P2Pt and P3Pt correspond to added drug, 0.5, 1 and 2% (w /w) of CDDP, P0Pt had zero concentration of cisplatin.

FTIR
We analyzed the infrared spectra together of the P0Pt to P3Pt bers (Fig. 1a), which share characteristic bands at 2150 and 1750 cm − 1 associated with the ester bonds of PLA.However, those bers with CDDP showed an increase in bands in the areas of 1640 − 1560 cm − 1 , and one special band in 1507 cm − 1 (Fig. 1b), which are associated with bonds of the amino groups of CDDP, demonstrating the presence of the pharmacological agent [40,41,42].

DSC
Using the DSC curves of the second heating cycle, the characteristic temperatures of semi-crystalline materials were obtained (Table 1), where variations in temperatures and enthalpies associated with the alteration in PLA chains due to the incorporation of CDDP can be observed but these modi cations are not associated with chemical bonding of Cisplatin -PLA.Glass transition temperature (Tg); crystallization temperature (Tc); melting temperature (Tm); enthalpy of crystallization (Hc); enthalpy of fusion (Hm).

TGA
The thermal behavior was also evaluated using the TGA technique (Fig. 1c), where a weight loss of 97% is observed for the P0Pt sample at 280 ° C, culminating in near 0% mass at up to 400 ° C.These results are similar to the temperatures reported by Qiu K. et al. [26].On the other hand, P0Pt bers (Tmax = 350 °C) had greater thermal stability than those containing CDDP (P1Pt Tmax = 341 ° C; P2Pt Tmax = 343 ° C; P3Pt Tmax = 341 ° C).Also, the latter presented residual mass percentages at 600 ° C (P1Pt = 1.3%;P2Pt = 1.7%;P3Pt = 3.39%) which can be attributed to the platinum content in the samples, since this metal has its point melting above 1400 ° C, and for the same reason, no other loss event is seen [43].The residual mass is above the theory weight (0.5, 1 and 2%) due a failure in the system parameters, not a fully homogenization of the polymeric solution with cisplatin could make some of the bers had a greater amount in different zones.

Morphology
In the micrographs (Fig. 2a, c, e, g) we can see that the bers are randomly arranged and have a smooth surface.In addition, in the P3Pt, an agglomerate of CDDP is observed outside its surface but contained within the composite.This may be due the polymer solution with which they were made was based on supersaturated DMF, therefore, only a part CDDP was solubilized [44].Likewise, we observed that when the drug was added to the bers, they decreased their size (Fig. 2b, d, f, h) with a greater frequency to tend to measure 1 µm in diameter.This is attributable to the platinum contained in the solution, since it has been shown to affect the surface charge of the solution, increasing the repulsive force of the magnetic eld under the applied voltage, causing the jet of expelled polymer to thin.Consequently, we obtained bers of less diameter than the P0Pt [45,46].The statistical analysis used for this assay was a mean with standard deviation (SD), with n = 10 for every sample, no statistical comparisons were made.

Degradation assay
Hydrolytic degradation in this type of composites occurs in two ways, homogeneous and heterogeneous.In the homogeneous one, the area of the bers does not change, but it swells and releases what is inside the composite, a fact that happened with the P3Pt bers (Fig. 3a), therefore, there was no change in weight or shape in the rst three months.This was contrary to what happened with the P0Pt to P2Pt bers, which degraded in a heterogeneous way, where surface erosion and deformation of the bers were observed.This loss of weight in uences the pH since the lactic acid oligomers separate, and over time, are hydrolyzed until decomposing into monomers, and due to this, we observed a greater decrease in the pH in the P0Pt bers (Fig. 3b ); in addition to the fact that, in a neutral or alkaline environment, lactic acid is in its ionized form due to its pKa of 3.84, which favors the breaking of the ester bonds [47].Therefore, the greater the degradation of the material, the greater the release of the drug contained in the bers, although it is well known that PLA does not degrade quickly but can maintain its initial weight in in vitro tests up to 6 months [48].

Hemocompatibility test
In this test (Fig. 3c), we observed that CDDP does not exert a damaging effect on the erythrocyte membrane in the time of 1 h, and the same happened for bers with and without drug (p < 0.05), which is similar to that observed by Da Silva et al. [49] where they evaluated PLA particles, and these were below the limit of 5% of allowance of the American Society of Testing and Materials (ASTM) [50].The statistical analysis used for this assay was an ANOVA test with post hoc of multiple comparisons, with n = 9 for every sample, the comparisons are made to evaluate the differences among the samples with and without CDDP, the alpha level in this test was 95% one tail and the P valor of 0.05.

In vitro cytotoxicity
We obtained the percentage of cell viability by means of an MTT assay (Fig. 3d).The HeLa cells were put in contact with the different bers and the free cisplatin (0.05 mg / mL) for 24 h.Subsequently, cell viability was determined.The P3Pt bers decreased cell viability by approximately 40%, in addition to having a greater amount of encapsulated CDDP, they presented agglomerates of drug outside the ber, resulting in what is known as explosive release, which is the release of the drug directly to the medium without degradation of the bers, to later enter a constant release stage.Furthermore, the diameter has an in uence, since, as has been observed, the larger the diameter of the drug-containing bers, the greater the impediment for its release, since the surface must rst be broken.Therefore, the P1Pt and P2Pt bers had less effect than the P3Pt [51].The P3Pt bers had a signi cant difference (p < 0.05) when compared to the free cisplatin, so we could assert that in the in vitro trials, the ber treatment works, although studies are needed to determine if it works as a treatment in situ in an in vivo model.The statistical analysis used for this assay was an ANOVA test with post hoc of Dunnet's, with n = 5 for every sample, the comparisons are made to evaluate the differences between the cytotoxicity level that was produced by the cisplatin release from the bers, the alpha level in this test was 95% one tail and the P valor of 0.05.

Conclusions
Poly (lactic acid) bers charged with CDDP were successfully made in our study.By adding the drug, the temperature characteristics of the material were modi ed.Fiber degradation and diameter decreased, compared to bers without drug.Those bers without drug were degraded in greater quantity at 5 months, for which their use is viable for extended times.The use of these bers turned out to be haemocompatible despite having the cytotoxic drug added, in contrast, cytotoxicity in HeLa cell culture was shown to have a signi cant difference with free cisplatin at the same drug concentration, indicating that its use may be viable for the treatment of some types of solid tumors.

Declarations Figures
The

Supplementary Files
This is a list of supplementary les associated with this preprint.Click to download.
infrared spectra of the P0Pt to P3Pt bers and Thermogram of the bers.a) FT-IR spectra of the bers.Which share characteristic bands at 2150 and 1750 cm -1 associated with the ester bonds of PLA.b) Enlargement of the area of 1660 -1500 cm -1 .Are associated with bonds of the amino groups of CDDP.Transmitance (%), Wave number (cm -1 ).C) Thermal behavior was also evaluated using the TGA technique.A weight loss of 97% is observed for the P0Pt sample from 280 ° C, culminating at up to 400 °C.Residual mass (%), Temperature (°C).

Table 1
DSC curves of the second heating cycle.Characteristic temperatures of the bers.