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Abstract
Interstitial photodynamic therapy (I-PDT), which utilizes optical fibers to deliver light for photosensitizer excitation and the elimination of penetration depth limitation, is a promising modality in the treatment of deeply seated tumors or thick tumors. Currently, the excitation domain of the optical fiber is extremely limited, restricting PDT performance. Here, we designed and fabricated a biocompatible polymer optical fiber (POF) with a strongly scattering spherical end (SSSE) for I-PDT applications, achieving an increased excitation domain and consequently excellent in vitro and in vivo therapeutical outcomes. The POF, which was drawn using a simple thermal drawing method, was made of polylactic acid, ensuring its superior biocompatibility. The excitation domains of POFs with different ends, including flat, spherical, conical, and strongly scattering spherical ends, were analyzed and compared. The SSSE was achieved by introducing nanopores into a spherical end, and was further optimized to achieve a large excitation domain with an even intensity distribution. The optimized POF enabled outstanding therapeutic performance of I-PDT in in vitro cancer cell ablation and in vivo anticancer therapy. All of its notable optical features, including low transmission/bending loss, superior biocompatibility, and a large excitation domain with an even intensity distribution, endow the POF with great potential for clinical I-PDT applications.
© 2023 Optica Publishing Group
Photodynamic therapy (PDT) utilizes photosensitizers (PSs) under irradiation with light of an appropriate wavelength to generate reactive oxygen species (ROS) to irreversibly destroy diseased tissues. Due to its notable advantages, including its non-invasiveness, few side effects, and high spatial resolution, PDT has been clinically applied to treat various cancers since its first approval for clinical use in 1993 [1]. Recently, various emerging PSs, including aggregation-induced emission fluorogens (AIEgens), carbon dots, metal-organic framework nanoparticles (MOF NPs), etc., have been designed and synthesized to improve the therapeutical outcomes of PDT [2–4]. However, the absorption of most existing PSs is located in the visible band, which has limited the penetration depth into biological tissue, significantly lowering PDT efficiency [5]. Considering that near-infrared (NIR) light has a deeper penetration depth as compared with visible light, novel NIR light excitation strategies, including two-photon and second-harmonic strategies, upconversion, etc., have been proposed to perform NIR-light-excited PDT and consequently improve the treatment depth and performance [6–8]. However, the penetration depth of NIR light is still limited to several millimeters, and NIR-light-excited PDT can’t meet the treatment requirements of deeply seated tumors or thick tumors.
Interstitial PDT (I-PDT) can excite PSs in deeply seated tumors or thick tumors by utilizing optical fibers to deliver excitation light, totally eliminating the penetration depth limitation. As such, I-PDT has been used for the treatment of various types of cancer, e.g., pancreatic cancer, brain cancer, etc. [9,10]. It is well known that the divergence angle of conventional optical fiber is small. Thus, I-PDT normally uses several optical fibers or one optical fiber with a diffusing part to achieve a similar excitation domain comparable in size to the tumor [11]. Recent studies suggested that diffuser fibers were more effective than flat-cut fibers in delivering the therapeutic light, and various efforts have been devoted to developing high-performance diffuser fibers [12,13]. Corning Incorporated developed Fibrance, a glass-based fiber optic cylindrical diffuser which can illuminate a fiber from 0.5 cm to 10 meters over a broad wavelength range [14]. Medlight developed a spherical distributor which is a silica-fiber-based catheter to cater to more diversified needs. However, these silica fibers suffered from fragility and biocompatibility issues when inserted into complex biological surroundings or implanted for the long term.
Polymer optical fibers (POFs), also referred to as plastic optical fibers, have some unique advantages as compared with conventional silica optical fibers, including high fracture toughness and high flexibility when bending [15]. Due to their design and fabrication flexibility, various types of POFs, including micro-structured POFs, fluorescent POFs, single-mode/multi-mode POFs, etc., have been developed for sensing, lighting, drug delivery, optogenetics, etc. [16–19]. Considering the diversity and flexibility of the preform material, POF could be a promising candidate for light delivery in I-PDT.
Here we designed and drew a new polymer optical fiber using polylactic acid (PLA) via a thermal drawing method for I-PDT applications. Various POF ends, including flat, spherical, conical, and strongly scattering spherical ends, were formed to study the divergence angle and excitation domain by experimental and theoretical analyses, the results of which matched well with each other. After optimizing the nanopores within the strongly scattering spherical end (SSSE) by changing the mass concentration of PLA in the modification solution, the POF with an SSSE achieved the largest excitation domain with an even intensity distribution among POFs mentioned above. The optimized POF possessed a low transmission/bending loss, superior biocompatibility, and a large excitation domain with an even intensity distribution, enabling excellent therapeutical outputs of I-PDT.
It is extremely difficult for conventional PDT to treat deep lesions due to the absorption and scattering of light in biological tissues. Interstitial PDT utilizes optical fibers, which are inserted into the target deep lesions to deliver light to activate a photosensitizer in deeply seated tumors or thick tumors, making I-PDT a promising modality in the treatment of locally advanced tumors. PLA-based POF with a highly scattering end was designed and fabricated for I-PDT studies since PLA is a biodegradable polymer with outstanding biocompatibility [20]. Moreover, the refractive index of PLA is higher than 1.45 in the visible light range [21], which is larger than that of most biological tissues [22]. The large refractive index of PLA ensured efficient confinement of light within the POF even without cladding when the POF was inserted into biological tissues. All these features make PLA a promising candidate for POF fabrication. As shown in Fig. 1(a), a thermal drawing method was used to fabricate the PLA-based POF (see the Supplement 1 for details). The diameter of the formed POF was about 500 ± 20 µm. The transmission loss of the POF was measured using the cut-back method. The measured transmission loss at 660 nm in air and tissue were 0.083 and 0.317 dB/cm [Figs. 1(b) and 1(c)], respectively. It could be calculated that the propagation length of 660 nm light within the POF in a biological sample could reach 9.6 cm (at which the light intensity was attenuated to 1/e of the incident intensity), enabling efficient I-PDT. Meanwhile, the practical transmission of visible light in different media via the POF was recorded by camera [Fig. 1(d)]. The scattered light from the POF in tissue was larger than that in air and water, which was consistent with the transmission loss measurement, but the light transmitted in tissue via the POF was strong enough for I-PDT excitation. Sometimes bending is inevitable in biological samples. Thus, the influence of bending on the POF was studied. The POF could be bent into 1-cm-diameter circles without fracture, and the bending loss of the POF was only about 20%, even when the bending radius reached 1 cm [Fig. 1(e)], enabling its utilization in complicated biological surroundings.
Fig. 1. (a) Schematic diagram of POF fabrication. The attenuation of the POF in (b) air and (c) chicken tissue. (d) Transmission features of POF when inserted into different media. (e) Bending loss of the POF.
Tumors are usually about a few centimeters in diameter. The excitation domain of I-PDT using one traditional optical fiber is much smaller than a tumor, limiting the therapeutical outcomes of I-PDT. Thus, it is extremely meaningful to expand the excitation domain of optical fibers. POFs with different ends [Fig. 2(a)], including flat, spherical, conical, and strongly scattering spherical ends, were fabricated for an excitation domain investigation. The forward divergences of the emitted light from POFs with different ends were simulated by the TracePro software and presented as polar iso-candela plots [Fig. 2(b)]. The emitted light from the POF with a flat end was distributed over only a small area, indicating a limited excitation domain. When the POF ended with a sphere, the divergence angle was further reduced instead of expanded, which could be ascribed to the sphere acting as a convex lens and focusing the emitted light beam. For the POF with a conical end, the divergence angle was efficiently expanded, but the emitted light was not evenly distributed, which could impair the I-PDT efficiency. When nanopores were introduced into the sphere to form an SSSE, the emitted light had a significantly expanded divergence angle and an even intensity distribution, making the POF with an SSSE an ideal device to achieve highly-efficient I-PDT. To further verify their divergence characteristics, these POFs with different ends were inserted into agarose gel, and the distributions of the laser light emitted from the POFs were recorded [Fig. 2(c)]. The POF with an SSSE enabled even excitation within a larger domain as compared with the other POFs, matching well with the theoretical analysis.
Fig. 2. (a) Four different fiber ends (flat, spherical, conical, and strongly scattering spherical ends from left to right). (b) Polar iso-candela diagram results for the forward divergence of light emitted from POFs with different ends. (c) Distributions of 532 nm laser light emitted from POFs with different ends.
Considering that the SSSE efficiently expanded the divergence angle and excitation domain, the corresponding nanopores were further optimized by varying the mass concentration of PLA in dichloromethane (DCM), which determines the nanoporous features in the modification process of the POF end [Fig. 3(a)]. The scattering coefficients of PLA films which were formed using DCM solutions with different mass concentrations of PLA (2%, 4%, 6%, 8%, and 12%), were measured using the Kubelka–Munk model. As shown in Fig. 3(b), as the PLA concentration increased, the scattering coefficient exponentially decreased. This may be because more PLA (or less DCM) would induce a more uniform film (with less nanopores) and consequently a smaller scattering coefficient. Meanwhile, a higher concentration of PLA would induce a more viscous DCM solution, leading to a larger diameter of the SSSE [Fig. 3(c)]. To analyze the influence of the mass concentration of PLA in the modification solution on the divergence angle, the distribution of the emitted light from the POF, including the forward-scattered and backward-scattered light, was simulated by the TracePro software and presented as polar iso-candela plots [Fig. 3(d)]. The scattering coefficients and end sizes measured above were used for modeling to obtain more accurate simulation results. All POFs with an SSSE exhibited large divergence angles in both the forward and backward directions. When the PLA concentration was low (2%, 4%, or 6%), many nanopores were introduced into the POF, which enhanced the scattering coefficient, leading to the even light distribution in both directions. Also, the forward and backward scattered light intensity was most balanced for the POF with a PLA concentration of 6%. Moreover, the average diameter of the SSSE made of 6% PLA was about 977 µm, ensuring that it can be used in a small incision suitable for in vivo applications. When the PLA concentration was high (8% or 12%), only a few nanopores were introduced into the POF. The resulting small scattering coefficient caused the scattered light to be mainly distributed within a small range in the forward direction, limiting the excitation domain and consequently the therapeutical outcome of I-PDT. Thus, the POF with an SSSE made of 6% PLA was selected for the following studies.
Fig. 3. (a) Schematic diagram of POF end modification. (b) Scattering coefficients of PLA films with different PLA mass concentrations (2%, 4%, 6%, 8%, and 12%). n = 3. (c) Diameters of POFs formed using solutions with different PLA concentrations (2%, 4%, 6%, 8%, and 12%). n = 5. Inset image shows the POF ends. (d) Polar iso-candela plots of the forward and backward light distributions emitted from POFs formed using solutions with different PLA concentrations (2%, 4%, 6%, 8%, and 12%).
Before performing I-PDT, the cytotoxicity of the POF was studied in vitro and in vivo. POFs with a flat end (FE) and an SSSE were soaked in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum at 37℃ for 48 h to get the leaching solution. After incubation with the leaching solution, the viability of HEK293 cells treated with leaching solution was evaluated by a Cell Counting Kit-8 (CCK-8) assay. As shown in Fig. 4(a), there was no significant difference between the control group and cells treated with leaching solution, demonstrating the good biocompatibility of the POF. Then, the in vivo biocompatibility of the POF was studied by subcutaneous implantation of the POF into the backs of ICR mice. On days 7, 14, and 21, the tissue around the implanted POF was collected and analyzed via hematoxylin and eosin (H&E) staining [Fig. 4(b)]. On day 7, leukocytosis could be observed, including lymphocytes, plasmacytes, and macrophages, around the implants, which is a normal immune response to a foreign object implanted into a body. On day 14, the inflammatory response was significantly decreased. On day 21, the implanted POF had fused well with its surrounding tissues, and the inflammatory response was further reduced. The POF exhibited superior in vitro and in vivo biocompatibility, enabling its long-term implantation in vivo.
Fig. 4. Biocompatibilities of the POFs. (a) In vitro cell viability of HEK293 cells treated with leaching solution of POFs for 24 h and 48 h. 1:1 indicates one POF in 1 mL leaching solution, and 2:1 indicates two POFs in 1 mL leaching solution. n = 4. (b) H&E staining of tissues around the implanted POF at different times. “F” indicates the area where the fiber resided. Scale bar: 200 µm.
To study in vitro cancer cell ablation using I-PDT, human prostate cancer (PC3) cells were incubated with chlorin e6 (Ce6, 2 µM) for 4 h, which was followed by irradiation by 660 nm light via POFs with an FE and an SSSE. The power density and irradiation time were 100 mW/cm2 and 5 min, respectively, keeping the same light dose for all groups. After being stained with calcein-AM and propidium iodide (PI), the treated PC3 cells were imaged by a fluorescence microscope (Fig. S1 in Supplement 1). After being irradiated via the POFs with an FE and an SSSE, all the PC3 cells within the irradiation domain were PI positive, indicating the good cancer cell ablation capability of I-PDT. Meanwhile, the PC3 cells treated with only light or Ce6 were calcein positive, eliminating any interference from light irradiation and Ce6. In order to compare the excitation domains, the whole culture dishes were imaged by an in vivo imaging system (IVIS). The PI-positive range induced by the POF with an SSSE was much larger than that induced by the POF with an FE, indicating that the POF with an SSSE would achieve highly efficient I-PDT (Fig. 5).
Fig. 5. Live/dead fluorescence images of PC3 cells treated with different irradiation conditions. Scale bar: 5 mm. Calcein channel [excitation (Ex): 488 nm, emission (Em): 505–525 nm], PI channel (Ex: 552 nm, Em: 605–625 nm), [Ce6] = 2 µM, [calcein-AM] = 2 µM, [PI] = 2 µM. FE light/I-PDT: light irradiation/I-PDT using the POF with an FE, SSSE light/I-PDT: light irradiation/I-PDT using the POF with an SSSE.
The POF with an SSSE exhibited superior biocompatibility, a uniform and large excitation domain, and an efficient in vitro I-PDT effect, making it a promising optical fiber for in vivo I-PDT. PC3 cells were subcutaneously injected into the dorsal area of nude male BALB/c mice to build a tumor-bearing model. The tumor-bearing mice were randomly divided into six groups (n = 4), including a control group (without any treatment), a PS group (with an intratumor injection of 5 mg/kg Ce6), an FE PDT/light group (with/without an intratumor injection of 5 mg/kg Ce6; 660 nm laser light was delivered by the POF with an FE, 60 mW, 20 min), and an SSSE PDT/light group (with/without an intratumor injection of 5 mg/kg Ce6; 660 nm laser light was delivered by the POF with an SSSE, 60 mW, 10 min). Here, “60 mW” refers to the power of the light scattered forward by the SSSE (measured with an optical power meter) which was about half of the total power of the scattered light [Fig. 3(d)]. The irradiation time for the POF with an SSSE was halved to ensure the same light dose. After the I-PDT treatment, the weight of the mice and the volume of tumors were recorded every other day for 14 days. There was no significant body weight loss in any of the groups within 14 days, as shown in Fig. 6(a). The tumor sizes of mice from all the groups increased, but the tumor-size increment rate of mice treated with I-PDT via the POF with a flat end was significantly lower than those in the control groups; moreover, the tumor size increment rate was further lowered by I-PDT using the POF with an SSSE [Fig. 6(b)]. The enhanced therapeutical outcomes of I-PDT can be ascribed to the even and larger excitation endowed by the SSSE. Then, all the mice were sacrificed on day 14, and the tumors were collected for further analysis. The tumor size in the SSSE I-PDT group was significantly smaller than those in the other groups [Figs. 6(c) and 6(d)], confirming the enhanced I-PDT performance endowed by the POF with an SSSE.
Fig. 6. (a) Body weight curves and (b) tumor volume curves for different groups of mice post-treatment (n = 4). (c) Excised tumor weights on day 14 post-treatment (n = 4). (d) Image of tumors excised on day 14 post-treatment.
In conclusion, we developed a new class of biocompatible POF by using a simple thermal drawing method, and further optimized the POF to form a strongly scattering spherical end by modifying the PLA concentration of the preform solution. The theoretical simulation and experimental demonstration, which matched well with each other, showed that the optimized POF had unique advantages, including a low transmission/bending loss, superior biocompatibility, and a large excitation domain with an even intensity distribution. The developed POF enabled I-PDT to achieve a large excitation domain and consequently excellent in vitro cancer cell ablation and in vivo therapeutical outcomes using a single POF, overcoming the penetration depth, biocompatibility, and excitation domain issues of traditional PDT/I-PDT. This study highlights the great potential that the development of a superior biocompatible POF with a strongly scattering-spherical end to advance the research field of single-POF-enabled I-PDT for further clinical research.
Funding
Ministry of Science and Technology of the People's Republic of China (2021YFF0502900); Shanghai Jiao Tong University (YG2022ZD005).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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