Magnetic nanofibers based bandage for skin cancer treatment: a non‐invasive hyperthermia therapy

Abstract Background The treatment of non‐melanoma skin cancer and deadliest malignant melanoma skin cancer are the fifth and ninth most expensive treatments in Medicare, respectively. Moreover, the recurrence of cancer after currently available therapies, that is, surgery or radiotherapy, reduces the patient's life expectancy. Aims In view of this, we fabricated magnetic nanofibrous mat‐based bandage to treat skin cancer non‐invasively using an external alternating current (AC) magnetic field induced hyperthermia. Methods The Fe3O4 nanoparticles incorporated polycaprolactone (PCL) fibers based bandages were fabricated using the electrospinning technique. The efficacy of the bandage was investigated in vitro using parental/doxorubicin hydrochloride (Dox)‐resistant HeLa cells and in vivo using BALB/c mouse model in the presence of an external AC magnetic field (AMF). Results The PCL‐Fe3O4 fibrous mat‐based bandages dissipate heat energy locally on the application of an external AMF and increase the surrounding temperature in a controlled way up to 45°C in a few mins. The in vitro study confirms the elevated temperature could kill parental and Dox‐resistant HeLa cells significantly. As the activity of Dox enhanced at a higher temperatures, more than 85% of parental HeLa cells were dead when cells incubated with Dox contained fibrous mat in the presence of AMF for 10 minutes. Further, we confirm the full recovery of chemically induced skin tumors on BALB/c mice within a month after five hyperthermic doses for 15 minutes. Also, there was no sign of inflammation and recurrence of cancer post‐therapy. Conclusion The present study confirms the PCL‐Fe3O4 nanofibrous based bandages are unique and compelling to treat skin cancer.

results in a large number of skin cancer cases per year. [3][4][5] Skin cancers are categorized in malignant melanoma (MM) and non-melanoma skin cancer (NMSC). MM is the deadliest cancer with a higher rate of metastasis. 6 NMSC is the most common cancer, which includes basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) skin cancers.
Besides sun or UV exposure, organ transplants or immunocompromised patients are found to be more susceptible to NMSC.
There are several therapies to treat skin cancers. Surgery is the primary option for skin cancer treatment, in which the infected skin lesion is removed with precision. In the advanced Mohs micrographic surgery (MMS), thin layers of skin cancer precisely excise and analyze under the microscope with color-coded margins by utilizing tangentially cut frozen-section histology. 7 Nevertheless, cosmetic defect or accompanying disease, 8 recurrence of cancer, 9 the opening of the wound, 10 wound infection, 11 and the requirement of graft for wound closure are the major challenges of the surgery. 12 If surgical excision is not possible due to the patient's medical condition or hard to operate the site, for example, the eyelids, inner and upper canthi, the ear, and nose, radiation therapy is considered for the treatment. 13,14 In radiation therapy, high energy radiations, such as X-rays and proton rays, are used to kill cancer-spreading cells. However, there is some safe limit of radiation doses one can have in their entire lifetime; therefore, a person cannot receive radiation therapy if that person already has taken maximum doses. 15 Radiation therapy can also increase the risk of subsequent skin cancer post-therapy. 5,16 Furthermore, topical chemotherapy involves the use of chemodrug locally to kill cancerous cells, which can be used to treat only NMSC. The major drawback of this therapy is that after a few doses, cancerous cells block chemodrugs induced apoptosis and become drug resistant. 17 This tendency of the cells makes the treatment difficult to kill tumor cells using chemodrugs further. The photodynamic therapy (PDT) involves the activation of a photosensitizing agent by light ranging from UVA to near-infrared wavelength. 18 The photosensitizer reaches an excited state that undergoes a reaction with ambient oxygen to create reactive oxygen species (ROS). These ROS then react with intracellular components and induce cell inactivation, followed by cell death. Since this therapy requires photosensitizing drug and light sources, consequently, it cannot be used for disseminated cancer. 19 Also, poor penetration of light in the case of melanoma cancer is the major limitation of this therapy. Another promising approach to treat cancer is hyperthermia therapy, which includes the delivery of heat energy to increase the surrounding temperature (40 C-45 C). It is reported that the hyperthermia induces endoplasmic reticulummediated apoptosis in melanoma and NMSC cells. 20 Further, hyperthermia can reverse the chemodrug resistance of the cells and enhance the drug activity. 21 The low doses of hyperthermia can enhance the anticancer drug efficacy by modifying responsible gene expression, for example, reducing the MDR1 gene expression, which increases drug sensitivity. 22 Therefore, the combination of hyperthermia and drug could enhance the efficacy of the treatment by reducing the cellular resistivity toward the anticancer drug.
The continuing advancement in technology provides ways to design smart systems to deliver heat to a localized area using radiofrequency radiation, laser ablation, microwaves, ultrasound waves, and magnetic field. The magnetic hyperthermia (MH) is based on the principle of delivering heat energy locally using magnetic materials with the application of an external alternating magnetic field (AMF). [23][24][25] The hyperthermia efficiency is dependent on the type of magnetic material, shape, size, and magnetic field strength. It is reported that the heating capabilities of a one-dimensional (rod shape) material is better than its zero-dimensional shapes (spherical particle). 26 Motoyama et al investigated the dependence of heating ability on the particle size and the AMF strength. 27 They found that the particles >1000 nm size with smaller specific-surface-area show a strong correlation of the heating capacity with the intensity of the AMF power (100-300 kHz and 2-32 kA/m) compared to particles~100 nm size, which show weaker cor- They reported apoptotic cellular death when cells were exposed to magnetic field of 17.0 kA/m and 183 kHz with Co-doped HFt for 30 minutes. Despite various studies of MNPs for hyperthermia therapy, there is no substantial evidence of the use of such systems clinically for the treatment of cancer due to their physical limitations; that is, agglomeration, non-uniform heating, and toxicity related to high dose or long term accumulation in the body. [33][34][35] The higher surface energy associated with MNPs leads to frequent agglomeration and the uneven distribution of agglomerated MNPs gives rise to nonuniform heating at the site of treatment. Additionally, the long-term accumulation of MNPs in the body induces toxicity, which could lead to an adverse side effect of the therapy. Herein, we are proposing magnetic nanofibrous based bandage to treat skin cancer using hyperthermia. The magnetic nanofibrous based bandages overcome the challenges associated with suspended MNPs and provide effective treatment for skin cancer in a short period. 36,37 Additionally, the localized heating reduces the associated side effects of the therapy and can be used easily in the case of multiple tumors.

| Fabrication of magnetic nanofibrous mat
The PCL and Fe 3 O 4 MNPs based fibers were fabricated using the electrospinning technique (E spin: Physics Equipment, Chennai, Tamil Nadu, India) as discussed previously. 23,27 Briefly, MNPs (3%, wt/vol) were mixed in PCL (15%, wt/vol), TFE, and DMSO solution using ultrasonicator before spinning. The volume ratio of TFE to DMSO was kept at 90:10. The distance between the syringe tip and the collector was 7 cm, and 20 kV voltage was applied during the electrospinning.
The morphology of the fibrous mat was analyzed using field emission scanning electron microscopy (FESEM). The SEM images were used to determine the diameter distribution of fibers through imageJ software. The hysteresis loss from the PCL-Fe 3 O 4 fibrous mat was recorded using a vibrating sample magnetometer (VSM: Lake Shore instrument, Westerville, OH, USA). Further, in the case of drug contained fibers, Dox was added in the PCL solution before electrospinning followed by ultrasonication. 37 The mats were suspended in phosphate buffer saline (PBS) and placed in the center of the induction coil (Ambrell Easy Heat solution) to measure the change in temperature. An AMF of 3.6 kA/m and frequency 236 kHz were applied for 10 minutes, and the change in temperature was measured using an alcohol thermometer. Further, the fibrous bandage was prepared using surgical tape and each bandage contained a 10 mg fibrous mat.

| Cell Culture
HeLa cells were obtained from American Type Culture Collection (ATCC), and the authenticity of the cells was confirmed using short tandem repeat (STR) analysis. A Dox-resistant variant of cervical cancer HeLa cell line was generated by stepwise selection with doxorubicin as mentioned above. 38 Briefly, HeLa cells were initially treated with 5 nM Dox. These treated cells were allowed to recover in the absence of Dox and resume proliferation after 1-week of treatment. A stable Dox-resistant HeLa cells were finally derived after 6 months of constant culturing in DMEM supplemented with 10% FBS and 1% antibiotic and antimycotic solution and maintained at 37 C with 5% CO 2 atmosphere with 90% humidity in an incubator (New Brunswick Galaxy 170S). It has been shown earlier that these cells are able to tol- The dorsal skin of all the mice was shaved using surgical clippers by gently restraining mice by the tail for no longer than 1 to 2 minutes.
One week after hair removal, 100 μL of DMBA (400 nmol) was applied to the shaved area, followed by restraining the mouse for an additional 5 to 10 seconds to allow the acetone solution to evaporate. Control mice received 0.2 mL acetone only. Mice were isolated in disposable biohazard caging for a week following the application of DMBA. After 1 week, 100-μL PMA was applied twice weekly to the DMBA-treated area. For around 90 days, all the mice's body weight was recorded at regular intervals to ensure that test and control mice maintain approximately equal rates of weight gain. The treatment was started with these BALB/c mice only when the palpable tumors attained a diameter of 5 mm or higher (as measured by vernier caliper) and those which persisted for 2 weeks or longer. The hair was removed carefully near the tumor before the onset of the treatment. The tumor lesion of all the TBH group mice was appropriately covered using a bandage and subjected to AMF to achieve a temperature of 45 C, whereas the mice of the TH group exposed to AMF without any bandage. The bandage temperature was maintained at 45 C for 15 minutes. This treatment was given to the tumor-induced mice for three consecutive days, followed by repetition on alternative days for two more sessions, that is, fifth and seventh day. After completion of the treatment, blood samples were collected to check on the liver and kidney functions. After 30 days of the treatment, the mice were sacrificed, and the skin samples were collected and analyzed by performing an hematoxylin and eosin (H&E) stain.

| Statistical analysis
All the experimental data obtained using the MTT assay and blood tests are expressed as mean ± standard deviation (M ± SD) and were analyzed by one-way analysis of variance (ANOVA; SPSS 16.0) for the calculation of significance level of the experimental data (n = 4). The differences were considered statistically significant at P ≤ .05.  Figure 1A). The diameter of fibers varies between 100 and 1000 nm, and its distribution can be described by normal Gaussian distribution, as shown in Figure 1B. The thickness of the deposited fibrous mat on the aluminum foil was controlled by deposition time and the flow rate of the solution. It is evident from the Figure 1C, the 10 mg fibrous mat suspended in 1 × PBS increased surrounding temperature by~22 C in 10 minutes in the presence of AMF (3.6 kA/m and frequency 236 kHz). Therefore, the 10 mg of the fibrous mat was pasted on the medical grade surgical tape in order to form a bandage, as shown in Figure 1D. Further, MH hysteresis loops infer magnetic nature of the system with the saturation magnetization (MS, emu/g) value 11 emu/g ( Figure 1E).

| Effect of hyperthermia on Dox-resistant HeLa cells
The Dox-resistant HeLa cells and parental HeLa cells were exposed to heat with or without Dox contained fibers using external AMF

| In vivo hyperthermia treatment
The efficacy of the magnetic fibrous mat-based bandages was tested for the treatment of skin cancer on the BALB/c mice using two-stage chemical carcinogenesis initiated by DMBA and promoted by PMA. Figure 3A shows the formation of the skin tumor after 5 to 6 weeks of chemical carcinogenesis. The nanofibrous bandage was placed on the tumor of TB group of mice and subjected to an external AMF  Figure 3B) for few minutes to achieve 45 C around the bandage and maintained this temperature for 15 minutes. In order to avoid the possibility of recurrence of cancer, the mice were given up to five heating cycles for 15 minutes (first 3 days, and then on fifth and seventh day). Figure 3C, there were no surroundings burning to the healthy skin near the tumor area and tumor completely disappeared with a small wound. The mice were healed fully after around 3 weeks of the treatment without further aids. As shown in Figure 3D, there is no sign of the inflammation, and hair growth was also restored in the treated area. The tumor volume in the case of untreated mice remained unchanged after suspending the application of the promoter TPA ( Figure 3E).

As shown in
In order to investigate the effect of heat on the underneath layers of skin, after 30 days of the treatment, the mice were sacrificed, and the tumor regions in treated and untreated mice were fixed in formalin and embedded in paraffin for histochemical analysis (Figure 4). The tumor region of the untreated mice in the UT group ( Figure 4B) and mice exposed to AMF without bandage in the TH group ( Figure 4C Table 1).

| DISCUSSION
Hyperthermia is an effective technique to treat cancer, yet the delivery of localized heat energy uniformly to kill cancerous cells is difficult to achieve. In the present study, the PCL-  Figure 1A and B). The fibrous mat dissipated heat energy and increased surrounding temperature when subjected to AMF ( Figure 1C). As shown in Figure 1E Figure 3A) were treated with hyperthermia at 45 C for 15 minutes using magnetic fibrous bandages ( Figure 1C). As shown in Figure 3B, the induced tumor near the tail of the mouse (TB group) was placed inside the induction coil after placing a bandage on the tumor. The frequency in the interval 50 kHz < f < 1 MHz and amplitude of the applied field (H < 15 kA/m) are recommended for the safety and comfort of the patients during treatment. 44 Furthermore, the number of heating cycles is also a critical parameter to determine the recurrence of tumors and formation of scar after treatment. The fewer heating cycles with low temperature can induce early cellular apoptosis (non-lethal damage) which can result in recurrence of cancer. 45 Moreover, intense AMF (>500 kHz and 15 kA/m) with one or two heating cycles can lead to severe burns to the tissue and forms a scar. Therefore, all the mice in the TB group were treated with up to five heating cycles for 15 minutes on first, second, third, fifth, and seventh day with an optimal and human tolerance limit of AMF, that is, 3.6 kA/m and frequency 236 kHz. After the treatment, the size of the tumor reduced significantly ( Figure 3C), and all the mice recovered completely after 2 weeks of the treatment ( Figure 3D). There was no reduction in the size of the tumor when mice were exposed to AMF without bandage (TH group), which confirms that the generation of heat through bandages killed the cells instead of external AMF. Additionally, all the mice showed a similar pattern of decrement in the tumor volume during treatment, while in other cases, the volume of the tumor did not change over time ( Figure 3E). As mentioned above, the generation of heat is dependent on the magnetic materials properties and external AMF strength. However, unlike the protein denature and DNA damage by direct heat flow to cells in vitro, the diffusion of heat in vivo depends on various other parameters, such as, the temperature of heat source, tissue thermal conductivity, tissue specific heat, and blood tissue density. 46 As presented in Figure 5, where the abnormal cells were grown in the full thickness of the epithelium and formed keratin pearls and lost hair follicles ( Figure 4B and C. Hou et al studied the efficacy of magnetic hydroxyapatite nanoparticles (mHAP) for the treatment of cancer with hyperthermia using a mouse model. 47 The mHAP NPs were injected at the tumor site, followed by the application of the AC magnetic field. They observed that the tumor shrank rapidly during treatment, and the animal recovered after 2 weeks of the treatment. However, they also observed elevated AST and ALP levels, which indicated a damaged

| CONCLUSIONS
The localized heating to treat skin cancer using a magnetic material is a compelling approach. We have shown that the magnetic fibrous mat- writing-review and editing. Shilpee Jain: Conceptualization; data curation; formal analysis; funding acquisition; methodology; project administration; resources; supervision; writing-original draft; writingreview and editing.

CONFLICT OF INTEREST
The authors have stated explicitly that there are no conflicts of interest in connection with this article.

ETHICAL APPROVAL
Animal protocols were approved by the Institutional Animal Ethics Committee (IAEC) and animals were maintained according to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines (a light/dark cycle of 12 hours, temperature 22 C ± 2 C).

DATA AVAILABILITY STATEMENT
Data are available on request from the authors.