When a cancer patient receives an infusion of cisplatin, the amount of drug that actually reaches the tumour tissue is surprisingly small: it is estimated that only 0.5-1% of the intravenously administered dose accumulates in the neoplastic mass, while the rest distributes throughout healthy tissues, particularly the liver, kidneys and lungs, generating the systemic toxicity that has always represented the most severe limitation of conventional chemotherapy. It is from this pharmacokinetic imbalance, even before any logic of innovation for its own sake, that the entire field of nanoparticle-based delivery of antitumour drugs originates, and within this landscape silk fibroin has carved out, in recent years, a space of growing scientific interest, to the point of being the subject of a recent and comprehensive review published in Communications Materials, which systematises synthesis strategies, physicochemical properties and oncological applications.
Fibroin as a biomedical platform
Bombyx mori fibroin is a structural protein that makes up approximately 75% by weight of the silk thread, the remaining 25% being represented by sericin, a water-soluble glycoprotein that must be removed during the degumming process because of its potential immunogenicity. What makes fibroin such a solid candidate for nanomedicine is not only its biocompatibility, already widely demonstrated by its established clinical use as a surgical suture material, but its intrinsic molecular architecture. The protein is composed of a heavy chain of approximately 390 kDa and a light chain of approximately 26 kDa, linked by disulphide bridges, with a strongly repetitive amino acid sequence dominated by glycine, alanine and serine, which favours the ordered packing of the chains into beta-sheet structures. It is precisely this capacity for conformational transition, from the metastable Silk I form to the thermodynamically stable Silk II form rich in antiparallel beta sheets, that gives fibroin that combination of mechanical robustness, controllable enzymatic degradability and formulation versatility that no other natural biopolymer replicates with the same effectiveness. Unlike non-biodegradable inorganic nanocarriers, such as gold nanoparticles, silica or carbon nanotubes, which tend to accumulate chronically in tissues, generating oxidative stress and genotoxicity, fibroin degrades into free peptides and amino acids, biocompatible and non-immunogenic, following a safety profile that places it, in terms of translational potential, alongside the albumin used in Abraxane nanoparticles, today the only true clinical standard among protein-based nanocarriers.
From cocoon to nanoparticle
The transformation of raw fibroin into a functional nanoparticle follows an articulated path that begins with degumming, typically carried out with sodium carbonate for its effectiveness and low cost, continues with dissolution of the fibre in chaotropic solvents such as lithium bromide or in organic solvents such as hexafluoroisopropanol, and concludes with dialysis and freeze-drying to obtain a regenerated fibroin ready for use. It is from this stock solution that the actual nanoparticles are built, through four main approaches that deserve to be distinguished because each produces nanoparticles with markedly different release characteristics, encapsulation efficiency and industrial scalability.
The desolvation method remains the most widespread: the gradual addition of the aqueous fibroin solution to a non-solvent such as ethanol, acetone or isopropanol induces the transition towards beta sheets and self-assembly into spherical particles, whose diameter can be modulated by acting on protein concentration, solvent addition rate, pH and temperature; as an indication, curcumin-loaded nanoparticles produced with this technique have shown diameters between 155 and 170 nanometres, with negligible cellular toxicity. Emulsification, which exploits water-in-oil or water-in-oil-in-water systems, allows smooth, spherical particles to be obtained but raises the issue of residual organic solvent, a non-trivial aspect when discussing clinical biocompatibility, so much so that some research groups have developed surfactant-free variants based on paraffin and water evaporation. Electrospraying, which uses high-voltage electrostatic forces to atomise the protein solution into a Taylor cone, has made it possible to produce cisplatin-loaded fibroin nanoparticles of just 59 nanometres in diameter without the use of any organic solvent, while preserving the drug's antitumour activity and reducing its side effects on healthy tissues. Finally, microfluidic technology today represents the most promising frontier for scale-up production: the millimetre-level control of flows between the fibroin solution and the precipitating agent, achieved through syringe pumps or pressure controllers, allows mixing times on the order of milliseconds and an extremely narrow size distribution, with polydispersity indices below 0.20 and size stability maintained for up to thirty days; in a recent study, nanoparticles produced via microfluidics with a cyclone mixer achieved encapsulation efficiencies of 82% for 5-fluorouracil, a result that concretely opens the way to production processes compliant with GMP standards.
Size, surface charge and behaviour in the bloodstream
Once the nanoparticle has been obtained, two physicochemical parameters determine its biological fate more than any other: size and surface charge. The optimal size window for fibroin nanoparticles lies between 50 and 300 nanometres, a range that directly influences biodistribution, tissue penetration, cellular internalisation and the prevailing endocytic pathway. Surface charge, measured as zeta potential, instead governs colloidal stability in the bloodstream: absolute values above ±30 mV are generally associated with good electrostatic repulsion between particles and therefore a lower tendency towards aggregation in physiological environments. Unmodified fibroin nanoparticles tend to carry a negative charge, but it is possible to reverse it by coating them with cationic polymers such as chitosan or polyethylenimine, an adjustment that not only improves their stability but also modifies their interaction with negatively charged cell membranes, favouring internalisation. A separate mention is deserved by PEGylation, which reduces recognition by the reticuloendothelial system and prolongs systemic circulation time, an essential requirement for the nanoparticle to have enough time to reach the tumour tissue before being cleared.
How the tumour microenvironment activates the nanoparticle
One of the most fascinating aspects of fibroin nanoparticles, and probably the one that most clearly distinguishes them from many synthetic carriers, is their intrinsic responsiveness to pathological stimuli, which does not necessarily require complex engineering. The tumour microenvironment is notoriously different from that of healthy tissues: more acidic pH, due to the accumulation of lactic acid linked to the glycolytic metabolism of neoplastic cells, elevated concentrations of reactive oxygen species and glutathione, and altered enzymatic activity. Studies conducted by David Kaplan's group have shown that acetone-precipitated fibroin nanoparticles display markedly pH-dependent drug release behaviour when the pH drops from 7.4, typical of blood, to 4.5, a value close to that of the intracellular endolysosomal environment. The mechanism is structural in nature: hydrogen ions, reactive oxygen species and glutathione destabilise the beta sheets and disulphide bridges of fibroin, loosening its packing and accelerating drug release exactly at the time and place it is needed. This behaviour can be further enhanced by introducing cleavable chemical bonds, such as diselenide, disulphide or boronate ester bridges, sensitive to the oxidative tumour environment, or hydrazone and ester bonds sensitive to pH, while maintaining stability under the physiological conditions of healthy tissues, a balance that is the true keystone of targeted delivery.
The EPR effect and active targeting strategies
The ability of fibroin nanoparticles to accumulate preferentially in tumour tissue is based first and foremost on the enhanced permeability and retention effect, the so-called EPR effect, a phenomenon whereby abnormal, hyperpermeable tumour blood vessels allow circulating nanoparticles to extravasate, which then accumulate there due to the impaired lymphatic drainage typical of solid neoplasms. Fibroin nanoparticles, with their size typically between 50 and 200 nanometres and hydrophilic surface, fit perfectly within the size window favourable to this passive accumulation mechanism. The most recent literature, however, calls for a less simplistic reading of EPR, acknowledging the contribution of active transport and retention processes that accompany the purely passive mechanism, and noting how the protein-based, relatively "soft" nature of fibroin orients it towards passive rather than active endothelial transcytosis pathways. To overcome the limitations and patient-specific variability of purely passive accumulation, the most effective strategy remains the combination of EPR with active targeting, achieved by functionalising the nanoparticle surface with ligands capable of recognising receptors overexpressed by tumour cells: folic acid, which binds to folate receptors often overexpressed in numerous epithelial neoplasms, the RGD peptide, which recognises integrins involved in tumour angiogenesis, or antibody fragments directed against tumour-specific antigens. A paradigmatic example is that of fibroin nanoparticles functionalised with cyclic RGD peptides and loaded with a genotoxic naphthalene diimide derivative: the surface decoration made it possible to selectively direct the highly cytotoxic drug towards tumour cells, reducing damage to healthy cells and containing systemic side effects.
From doxorubicin to multidrug resistance
The range of chemotherapeutic agents successfully delivered through fibroin nanoparticles is now broad and includes doxorubicin, paclitaxel, cisplatin, curcumin, auranofin and numerous other agents, both hydrophobic and hydrophilic. The case of doxorubicin is probably the most instructive, as it illustrates well how the combination of responsive release and active targeting can translate into a real therapeutic advantage against multidrug resistance, one of the most feared problems in clinical oncology. In a study that combined multi-responsive fibroin nanoparticles, capable of simultaneously releasing doxorubicin and a hypoxia-inducible factor inhibitor, with folic acid surface functionalisation for active targeting, the minimum effective dose of the delivered drug against resistant breast cancer cells (MCF-7/ADR) was found to be approximately twenty-six times lower than that of free doxorubicin, a result reflecting not only more efficient intratumoural accumulation but also the system's ability to bypass the efflux mechanisms responsible for chemoresistance. Similar folic acid functionalisation strategies, achieved through covalent conjugation between fibroin's amine groups and the ligand's carboxyl groups, have enabled selective internalisation by tumour cells with overexpressed folate receptors, improving chemotherapeutic efficacy while simultaneously reducing the adverse effects associated with doxorubicin. On the cisplatin front, electrospray production of particularly small nanoparticles, around 59 nanometres, has made it possible to preserve cytotoxic activity against tumour cells while minimising damage to healthy tissues, a result of particular relevance given that nephrotoxicity remains one of the main limiting factors in the clinical use of this drug. Auranofin too, a gold compound originally developed for rheumatoid arthritis and now repositioned in experimental oncology, has been encapsulated in fibroin nanoparticles for the treatment of colorectal cancer, as has curcumin, whose historical limitation of extremely poor oral bioavailability finds in protein-based nanodelivery one of the most widely studied solutions in the literature.
