Laminarin-Based Nanoparticles Deliver Protoporphyrin IX for Enhanced Photodynamic Therapy: Cellular Uptake, ROS Generation, and In Vivo Efficacy

Abstract

Laminarin conjugate–derived amphiphilic micelles (HLDM) were engineered to encapsulate the hydrophobic photosensitizer protoporphyrin IX (Pp IX) for breast cancer photodynamic therapy (PDT). The resulting micelles, ~149 nm in diameter, exhibited dual pH‑ and redox‑responsive release, improving aqueous solubility and biocompatibility. In vitro studies with MCF‑7 cells revealed efficient cellular uptake, light‑induced reactive oxygen species (ROS) generation, and apoptosis marked by nuclear damage. In a nude‑mouse xenograft model, Pp IX‑loaded HLDM micelles combined with 630 nm irradiation produced a pronounced tumor‑growth inhibition with no observable systemic toxicity. These findings demonstrate that a laminarin‑based nanoplatform can markedly enhance Pp IX delivery and PDT efficacy in vitro and in vivo.

Introduction

Photodynamic therapy (PDT) leverages a photosensitizer, light, and molecular oxygen to produce ROS that selectively kill tumor cells while sparing normal tissue. It has emerged as a minimally invasive option for diverse malignancies—including breast, liver, lung, melanoma, and skin cancers—yet its clinical adoption has been constrained by limited tumor accumulation of conventional photosensitizers.

Protoporphyrin IX (Pp IX) is a potent but hydrophobic photosensitizer that often aggregates in aqueous media, reducing its therapeutic window. Marine polysaccharide laminarin, known for its biocompatibility and intrinsic antitumor activity, offers a hydrophilic shell that can stabilize hydrophobic drugs and confer tumor‑targeting properties through pH and redox responsiveness.

We report the synthesis of a dual pH/redox‑responsive hematin‑laminarin‑dithiodipropionic acid‑MGK (HLDM) micelle system that encapsulates Pp IX, aiming to improve solubility, enhance tumor accumulation, and maximize ROS‑mediated cytotoxicity.

Methods

Materials

Laminarin (Sigma‑Aldrich), DMSO, L‑glutathione, Hoechst 33342, DMEM, FBS, ROS assay kit (Beyotime), H&E reagents, Pp IX (Aladdin), and all other reagents were of analytical grade. Human breast cancer MCF‑7 cells were obtained from Yantai University, and female nude mice (14–18 g) were sourced from Beijing Vital River.

Synthesis and Characterization of HLDM

HLDM was synthesized via sequential acylation of dithiodipropionic acid with MGK, followed by EDC/DMAP‑mediated esterification with laminarin. The product was verified by ^1H‑NMR, IR, and UV‑vis spectroscopy.

Preparation of Pp IX‑Loaded HLDM Micelles

HLDM and Pp IX were co‑dissolved in organic solvent, then dialyzed against deionized water (MWCO 2000 Da) under stirring (600 rpm). Micelles were collected at room temperature.

Characterization

Particle size and zeta potential were measured with a Beckman Coulter analyzer; morphology was examined by TEM (Hitachi H‑600). Encapsulation efficiency (EE) and drug loading (DL) were calculated from UV‑vis absorbance at 630 nm.

Cell Culture and Uptake Studies

MCF‑7 cells were cultured in DMEM/FBS. For uptake, cells were incubated with free Pp IX, LH micelles, or HLDM micelles (20 µg mL^−1) for 1–4 h. Fluorescence was observed by inverted microscopy (Nikon Eclipse E400).

Subcellular Localization

After 4 h incubation, cells were fixed, counterstained with Hoechst 33342, and imaged to determine Pp IX distribution.

ROS Detection

MCF‑7 cells were treated with formulations (20 µg mL^−1) for 2 h, irradiated at 630 nm for 30 min, then incubated with DCFH‑DA (10 µmol L^−1). Fluorescence (exc 488 nm, em 525 nm) indicated ROS production.

Phototoxicity and Viability Assay

Cells were seeded in 96‑well plates, exposed to varying concentrations (1–10 µg mL^−1) of each formulation, irradiated (630 nm, 30 min), and incubated for 24 h. MTT (5 mg mL^−1) was added, and absorbance at 490 nm measured to calculate viability.

Nuclear Morphology

After 4 h treatment, cells were fixed, stained with nuclear dye, and imaged to assess chromatin condensation.

In Vivo Efficacy and Safety

MCF‑7 cells (1.5 × 10^6) were subcutaneously injected into nude mice. Once tumors reached 70–100 mm^3, mice received one of five treatments: saline, free Pp IX (5 mg kg^−1), HLDM micelles (5 mg kg^−1 Pp IX equivalents), free Pp IX + 630 nm light, or HLDM micelles + 630 nm light. Tumor volume and body weight were monitored every other day; mice were euthanized on day 20 for histology (H&E).

Statistical Analysis

Data are expressed as mean ± SD (n = 3). Differences were assessed by one‑way ANOVA, with *P < 0.05 considered significant.

Results and Discussion

HLDM Characterization

^1H‑NMR confirmed the successful conjugation of MGK, dithiodipropionic acid, hematin, and laminarin, with characteristic peaks at δ 0.8, 2.8, 6.5, and 3–4 ppm. IR spectra displayed the expected ester carbonyl band, and UV‑vis showed a distinct 580 nm absorption corresponding to hematin within HLDM.

Micelle Properties

Dynamic light scattering revealed an average diameter of 149.3 ± 35 nm and a near‑neutral zeta potential. TEM images confirmed spherical morphology with occasional aggregation at higher concentrations. EE and DL were 68 % and 12 %, respectively, indicating efficient loading.

Cellular Uptake

Fluorescence imaging demonstrated time‑ and dose‑dependent accumulation of Pp IX within MCF‑7 cells, with HLDM micelles showing markedly higher uptake than free Pp IX or LH micelles. The enhanced uptake is attributed to the pH/redox‑sensitive release mechanism.

Subcellular Distribution

Co‑localization studies revealed predominant cytoplasmic and mitochondrial accumulation of Pp IX delivered by HLDM micelles, consistent with the known organelle‑targeting of Pp IX.

ROS Generation

DCFH‑DA fluorescence was significantly higher in cells treated with HLDM micelles plus light compared to free Pp IX, confirming robust ROS production only under illumination.

Phototoxicity

Under light irradiation, HLDM micelles achieved >80 % cell kill at 5 µg mL^−1, whereas free Pp IX required >10 µg mL^−1 for comparable effect. In the dark, all formulations exhibited minimal cytotoxicity.

Nuclear Morphology

Pp IX‑loaded HLDM micelles induced dose‑dependent chromatin condensation and nuclear fragmentation, indicative of apoptosis.

In Vivo Efficacy

Only the HLDM micelle group receiving 630 nm light showed significant tumor growth inhibition (≈ 70 % reduction at day 20). Free Pp IX with light produced modest suppression, while saline and non‑irradiated groups continued to grow. Body weight remained stable across all groups, and H&E staining revealed extensive necrosis in the HLDM + light cohort.

Conclusions

We have engineered a laminarin‑based, dual pH/redox‑responsive micelle that efficiently encapsulates Pp IX, enhances its solubility, and facilitates tumor‑specific release. The system delivers potent ROS‑mediated phototoxicity in vitro and achieves pronounced antitumor activity in vivo without systemic toxicity. These results position HLDM micelles as a promising platform for future photodynamic therapy of breast and other solid tumors.