Localized cancer photodynamic therapy approach based on core–shell electrospun nanofibers

Tsumugi
She is a second-year researcher in the Nanofiber Division of MECC.
Her hobbies include reading science magazines and the latest
research papers, as well as karate. Her special skill is speaking Chinese.

<Background and Purpose>
The fourth largest number of cervical cancers among women. Treatment includes surgery, radiation therapy, and chemotherapy, but side effects are common and efficacy is limited. Therefore, alternative treatments such as photodynamic therapy (PDT) are being considered to minimize side effects and prevent recurrence.

PDT is a treatment method based on the principle that various biomolecules are oxidized and the target tissue is irreversibly destroyed by generated singlet oxygen. Photoactive molecules called photosensitizers (PS) are absorbed in the cancerous cells, and PS is activated by irradiating the target site with a specified wavelength.

Porphyrins (Por) and their derivatives are commonly used in PS. While Por has advantages such as high singlet oxygen-quantum yield, minimal dark toxicity, and rapid removal from the body, it also has several drawbacks, such as a large amount of dose required for full-body administration to achieve a stable effectiveness, leading to prolonged light sensitivity, and the possibility of compound elimination by targeting the localized area of the tumour.

We intend to incorporate PS into the Local Drug Delivery Systems (DDS) to solve these shortcomings. Implantation of a local DDS at or near the tumour site allows direct delivery of PS to the target site, increasing treatment effectiveness, reducing side effects on surrounding healthy cells, the possibility of sustained PS release, protecting against degradation of PS prior to reaching the target, preventing PS agglomeration, and increasing the frequency of drug use.

Electrospinning technology is a highly versatile technology that can easily fabricate nanofibers and can adapt to various requirements. It is also attracting attention in developing nanofibers that function as a local DDS. Electrospinning nanofibers stand out as a basis for DDS in cancer therapy because of their high contrasting surface area, enabling target-specific drug delivery, simultaneous encapsulation of multiple pharmaceuticals, and control of drug profiles in terms of efficient drug encapsulation, a wide range of material-to-therapy options, and so on. It also has a structure similar to extracellular matrices, and is thin, small, and flexible so it can enter into small gaps. In cervical cancers, it is very promising because it is accessible from the vaginal canal and DDS can be implanted in or around the tumour cells and irradiated. Phototherapy nanofibers can also be diagnosed and treated at the same time.

Core-shell nanofibers can be fabricated using coaxial electrospinning techniques to simultaneously use two different polymer solutions. By combining the mechanical strength of these two polymers with the biological activity of natural polymers, it is an efficient way to develop DDS. The core acts as a single bioactive molecule, and the shell acts as a protective layer to prevent its degradation and premature release.

Nanofibers as a local DDS and PDT benefits as a cancers treatment are well established, but combined treatments have not yet been fully examined. The authors have therefore conducted studies aimed at developing core-shell electrospinning nanofibers loaded with Por, a PS agent that acts as a local DDS of PDT.

<Materials>
Polyvinyl alcohol (PVA), gelatine (Gel), synthetic Por

<Method>
After synthesizing Por and evaluating the singlet-oxygen and photodynamic effects, we incorporated Por into the core-shell electrospinning nanofibers using electrospinning techniques. At this time, PVA is used for the core layer and Gel is used for the shell layer. The nanofibers produced by electrospinning were then characterized to evaluate their drug release profiles in comparison under dark conditions and under radiation for the tumour cell lines and non-tumour cell lines.

Results

・Singlet Oxygen Generation and Cytotoxicity of Por in Tumor and Non-Tumor Cell Lines

We synthesized 5,10,15,20-tetrakis(4-carboxymethylthio-2,3,5,6-tetrafluorophenyl)porphyrin (TPPF₁₆[S-CH₂-COOH]₄) from an experimental procedure already published.

We have confirmed that the singlet oxygen-generating ability of TPPF₁₆[S-CH₂-COOH]₄ is comparable to that of tetraphenylporphyrin (TPP). We will use it in the following tests.

Next, we assessed the cytotoxicity of the cells against tumours and non-tumours. Por level (GI₅₀) required to inhibit cell growth by 50% was very low under illumination when compared to dark conditions. This demonstrates that PS is highly cytotoxic to cancerous cells when irradiated by light. It requires very low levels of Por to inhibit the growth of cancerous cells. GI₅₀ in non-tumour cells also indicated that high levels of Por were needed under both dark and light-irradiated conditions, confirming that Por was more cytotoxic to tumour cells than to non-tumour cells.

In addition, Por showed 11 times more phototoxicity than dark conditions for tumour cells under light-irradiated conditions, and only 2 times for non-tumour cells, indicating that phototoxicity of Por was mitigated in non-tumour cells.

・Integration of Por into PVA-Gelatin Core Shell Nanofibers

PVA-gelatin core shell nanofibers were fabricated using a coaxial electrospinning method. The coaxial accessory produced the one of the image.

Sofia M. Costa, Leandro M. O. Lourenço, Ricardo C. Calhelhacd, et al. : (quoted from Localized cancer photodynamic therapy approach based on core–shell electrospun nanofibers, Materials Advances, Issue 16, 6489-6500,21th August 2024.)

The fabricated nanofibers were characterized by FESEM, TEM, TGA, ATR-FTIR and were found to be smooth and defect-free nanofibers, containing both PVA and gelatine polymers.

We fabricated nanofibers with four Por levels, taking into account GI₅₀ when incorporating Por into nanofibers, and evaluated and analyzed them.

When Por was added to PVA, the viscosity/conductivity of the solutions increased according to Por density. It is thought that the incorporation of Por increased the density of electric charges, increased the conductivity, and increased the elongation of the ejected jet. As a result, the decrease in fiber diameter is accelerated.

The presence of Por in nanofibers was confirmed by GSDR. The characteristic absorption band of PS was found, and the obtained spectrum was very similar to that of Por alone, confirming the presence of Por in the nanofiber, and increasing Por content increased the strength of the band.

Por profile in the fiber was evaluated by CLSM. The nanofibers were excited with 405 nm and the emitted fluoresces were detected with 500-699 nm. In PVA-gelatin nanofibers, no fluorescence was detected when excited by 405nm, whereas red fluorescence was detected in PVA-gelatin + Por nanofibers loaded with Por. The continuity of this fluorescent light along the fiber indicates that Por is distributed throughout without aggregation.

Electrospinning nanofibers also feature high carrying capacity and high encapsulation efficiency, making them a very attractive platform for drug delivery. The loading capacity and encapsulation efficiencies of PVA-gelatin+ Por nanofibers were measured and both were high-value, demonstrating their suitability for DDS.

From GSDR, CLSM, we can confirm that PS was successfully incorporated into the electrospinning nanofiber. In addition, Por encapsulation within the fiber does not affect its properties, allowing real-time treatment monitoring parameters and treatment response assessment with inherent fluorescent properties, and allowing subsequent treatment programme planning and coordination.

・Por emission profiles from core-shell nanofibers

The release of Por was observed by UV-Vis spectroscopy when the fabricated nanofibers were immersed in solutions close to the vaginal milieu and agitated. Absorption spectra and drug release profiles obtained using PVA-gelatin nanofibers and PVA-gelatin plus Por nanofibers were represented graphically.

Sofia M. Costa, Leandro M. O. Lourenço, Ricardo C. Calhelhacd, et al. : (quoted from Localized cancer photodynamic therapy approach based on core–shell electrospun nanofibers, Materials Advances, Issue 16, 6489-6500,21th August 2024.)

Only the spectrum of PVA-gelatin plus Por nanofibers showed a typical absorption-band appearance, indicating the release of Por over time.

Several factors contribute to the drug release profile from nanofibers, including the physical properties of the drug, the structural properties of the polymer matrix, and the release conditions. With core-shell nanofibers, the encapsulated molecules need to pass through the matrix in both the core and shell layers, allowing for more sustained drug release.

In addition, Store bands were zoomed to evaluate Por emission, and the presence of the bands was observed from 1 hour to 8 hours. This may expose PVA to a portion of the leaky fiber surface during electrospinning, which promotes the rapid release of Por. Por was then released gradually over time. This result may be related to the degradation of the core-shell structure that allowed the diffusion of molecules from the core of the nanofibers into the solution.

We also plotted Por level over time. We observed the fast drug release file during the first few hours and confirmed that it was released continuously and gradually thereafter. This may indicate that the presence of the shell-layer is retarding the spread of Por.

These findings indicate that Por is continuously released after the first early release, confirming that PVA-gelatin+ Por nanofibers have a continuous and sequential DDS function for cervical cancer therapy.

・Cell Toxicity Test of Electrospinning Nanofiber in Tumor and Non-Tumor Cell Lines

To evaluate the capability of Por to act as a PS when incorporated into nanofiber membranes, we performed cytotoxicity studies on PVA-gelatin nanofibers and PVA-gelatin plus Por nanofibers. After the cells were incubated with nanofibers and the membranes were removed, the cells were irradiated with LED lamps. The results are shown in the table below.

Sofia M. Costa, Leandro M. O. Lourenço, Ricardo C. Calhelhacd, et al. : (quoted from Localized cancer photodynamic therapy approach based on core–shell electrospun nanofibers, Materials Advances, Issue 16, 6489-6500,21th August 2024.)

PVA-gelatin nanofibers exhibited minimal cytotoxicity in both dark and light-irradiation conditions. Numerical values under both conditions are also similar, and it can be confirmed that the characteristics under irradiation are not shown.

PVA-gelatin + Por nanofibers accelerated the inhibition of tumour cells in dark and light-irradiated conditions, and the inhibition rate was proportional to Por level. Especially, the inhibition rate under light irradiation is much higher than that under dark conditions, indicating that phototoxicity remains even after Por is incorporated into the nanofiber.

In addition, minimal Por additions have been achieved to avoid darkness toxicity and skin photosensitivity.

In the present experiment, the nanofibers are removed after 24 hours of contact with the cells, so the observed phototoxic effect is due to the molecules released after that period. The release of Por from nanofibers has been confirmed by drug release tests for at least 216 hours, suggesting that nanofibers can be applied for long-term release and that repeated dosing of drug can be avoided.

PVA-gelatin nanofibers exhibited very low toxicity to non-tumour cells, exhibiting no specific effect on light, cell growth inhibition was consistently lower than that observed in tumour cells, and the differences in the values obtained under dark and light irradiation were also fairly low, suggesting that there is a selective phototoxicity effect in tumour cell lines.

These findings indicated that the photodynamic effect of Por molecules was not affected by their incorporation into nanofibers, and could be a substrate for carrying photoactive molecules that could destroy tumour cells under illumination. These fibrous platforms have selective phototoxicity in tumour cell lines, highlighting their suitability and potential for PDT approach of treating tumour cells while maintaining safety against non-tumour cells.

<Summary>
We have developed a photoresponsive core-shell nanofiber by utilizing coaxial electrospinning technology. This nanofiber is made using PVA and gelatine, which are biodegradable polymers. The cores contain differently concentrated Por.

Synthesized Por produced singlet oxygens and showed much higher cytotoxicity to the tumour cells under illumination, indicating that they are promising candidates to function as PS for PDT. Por could be incorporated without affecting the morphology of the nanofiber, and was distributed throughout the nanofiber.

These fibrous nanoplatforms exhibit high Por loading and high encapsulation efficiency. Por release from the nanofibers was continuously released for 9 days. The initial stage was an early release followed by a slow, sustained release. This is thought to be due to the core-shell structure of the nanofibers.

PVA-gelatine + Por nanofibers showed a photodynamic effect. The higher percentage of inhibiting the growth of tumour cells under light irradiation than under dark conditions confirms that PVA-gelatin+ Por nanofibers are capable of inhibiting the growth of tumour cells when exposed to radiation. Furthermore, the tumour cell lines exhibited a selective phototoxic effect compared to non-tumour cell lines. In this study, nanofibers incorporating PS have shown great potential to serve as a localized DDS to PDT of cancers, particularly to cervical cancers, and to enable the sequential release of Por.

Our company produces various nanofiber samples. If you have any materials you would like to test or any questions regarding spinning, please feel free to contact us.

<Localized cancer photodynamic therapy approach based on core–shell electrospun nanofibers>
Localized Cancer Photodynamic Therapy Approach Based on Core-Shell Electrospinning Nanofibers
Sofia M. Costa, Leandro M. O. Lourenço, Ricardo C. Calhelhacd, Isabel Calejo, Cristina C. Barrias, Raul Fangueiro and Diana P. Ferreira
(Centre for Textile Science and Technology (2C2T), University of Minho, 4800-058 Guimarães, Portugal.) (https://pubs.rsc.org/en/content/articlelanding/2024/ma/d4ma00647j)