Targeting Peptides: The New Generation of Targeted Drug Delivery Systems.

Peptides can act as targeting molecules, analogously to oligonucleotide aptamers and antibodies. They are particularly efficient in terms of production and stability in physiological environments; in recent years, they have been increasingly studied as targeting agents for several diseases, from tumors to central nervous system disorders, also thanks to the ability of some of them to cross the blood-brain barrier. In this review, we will describe the techniques employed for their experimental and in silico design, as well as their possible applications. We will also discuss advancements in their formulation and chemical modifications that make them even more stable and effective. Finally, we will discuss how their use could effectively help to overcome various physiological problems and improve existing treatments.

Hybrid nano-architectures loaded with metal complexes for the co-chemotherapy of head and neck carcinomas.

Head and neck squamous cell carcinomas (HNSCCs) are a complex group of malignancies that affect different body sites pertaining to the oral cavity, pharynx and larynx. Current chemotherapy relies on platinum complexes, the major exponent being cisplatin, which exert severe side effects that can negatively affect prognosis. For this reason, other metal complexes with less severe side effects are being investigated as alternatives or adjuvants to platinum complexes. In this context, exploiting (supra)additive effects by the concurrent administration of cisplatin and emerging metal complexes is a promising research strategy that may lead to effective cancer management with reduced adverse reactions. Here, the combined action of cisplatin and a ruthenium(II) η6-arene compound (RuCy), both as free molecules and loaded into hybrid nano-architectures (NAs), has been assessed on HPV-negative HNSCC models of increasing complexity: 2D cell cultures, 3D multicellular tumor spheroids, and chorioallantoic membranes (CAMs). Two new NAs have been established to explore all the delivery combinations and compare their ability to enhance the efficacy of cisplatin in the treatment of HNSCCs. A significant supra-additive effect has been observed in both 2D and 3D models by one combination of treatments, suggesting that cisplatin is particularly effective when loaded on NAs, whereas RuCy performs better when administered as a free compound. Overall, this work paves the way for the establishment of the next co-chemotherapeutic approaches for the management of HNSCCs.

Pro-apoptotic and size-reducing effects of protein corona-modulating nano-architectures enclosing platinum prodrug in in vivo oral carcinoma.

The selective and localized delivery of active agents to neoplasms is crucial to enhance the chemotherapeutic efficacy while reducing the associated side effects. The encapsulation of chemotherapeutics in nanoparticles decorated with targeting agents is a strategy of special interest to improve drug delivery. However, serum protein adsorption often compromises the in vivo efficiency of targeting agents, leading to protein corona formation that interferes with the targeting process. Here, the enhanced efficacy of hybrid nano-architectures enclosing a platinum prodrug and decorated with a customized peptide (NAs-cisPt-Tf2) is demonstrated by employing alternative in vivo models of oral carcinoma. The peptide binds to transferrin and modulates the protein corona formation on NAs-cisPt-Tf2, supporting the identification of its receptor. Optimized chorioallantoic membrane cancer models (CAMs) enabled a thorough assessment of the tumor-suppressing effect of NAs-cisPt-Tf2 as well as the quantitative evaluation of angiogenesis and cell cycle associated mechanisms. The treatment strategy resulted in a significant tumor volume reduction coupled with anti-angiogenic and pro-apoptotic effects inferred from the downregulation of the vascular endothelial growth factor gene and increased expression of cleaved caspase-3. Overall, this study provides a potentially effective tumor-targeted approach for a non-invasive management of oral carcinoma.

Lysosomal Proteomics Links Disturbances in Lipid Homeostasis and Sphingolipid Metabolism to CLN5 Disease.

CLN5 disease (MIM: 256731) represents a rare late-infantile form of neuronal ceroid lipofuscinosis (NCL), caused by mutations in the CLN5 gene that encodes the CLN5 protein (CLN5p), whose physiological roles stay unanswered. No cure is currently available for CLN5 patients and the opportunities for therapies are lagging. The role of lysosomes in the neuro-pathophysiology of CLN5 disease represents an important topic since lysosomal proteins are directly involved in the primary mechanisms of neuronal injury occurring in various NCL forms. We developed and implemented a lysosome-focused, label-free quantitative proteomics approach, followed by functional validations in both CLN5-knockout neuronal-like cell lines and Cln5-/- mice, to unravel affected pathways and modifying factors involved in this disease scenario. Our results revealed a key role of CLN5p in lipid homeostasis and sphingolipid metabolism and highlighted mutual NCL biomarkers scored with high lysosomal confidence. A newly generated cln5 knockdown zebrafish model recapitulated most of the pathological features seen in NCL disease. To translate the findings from in-vitro and preclinical models to patients, we evaluated whether two FDA-approved drugs promoting autophagy via TFEB activation or inhibition of the glucosylceramide synthase could modulate in-vitro ROS and lipid overproduction, as well as alter the locomotor phenotype in zebrafish. In summary, our data advance the general understanding of disease mechanisms and modifying factors in CLN5 disease, which are recurring in other NCL forms, also stimulating new pharmacological treatments.

Ruthenium arene complexes in the treatment of 3D models of head and neck squamous cell carcinomas.

Current chemotherapy for head and neck squamous cell carcinomas (HNSCCs) are based on cisplatin, which is usually associated to severe side effects. In general, the exploration for metal-based alternatives to cisplatin has resulted in the development of a series of ruthenium complexes that are able to produce a safe therapeutic action against some neoplasms, among which are lung and ovarian cancers. Here, we evaluate the efficacy of well defined, easily available and robust ruthenium(II) η6-arene compounds on 3D models of HNSCCs with or without human papillomavirus (HPV) infection and compare their effects to the state-of-the-art RAPTA-C, a promising ruthenium compound with known anti-cancer activity. One of the compounds induces a significant therapeutic action especially on HPV negative carcinoma. Besides viability and repopulation evaluations, we performed quantitative analysis of the internalized Ru compounds to further validate our findings and elucidate the possible mechanisms of action. These results show that Ru arene compounds represent a promising alternative for the treatment of HNSCCs and pave the way for the composition of innovative (co)therapies.

Combined chemo-photothermal treatment of three-dimensional head and neck squamous cell carcinomas by gold nano-architectures.

Synergistic combined treatments are currently practiced in clinics for the management of several neoplasms. While surgery, radiotherapy, and chemotherapy remain as the standards of care for monomodal and co-treatments, emerging modalities like hyperthermia (HT) demonstrate promising features as (neo)adjuvant, particularly for recurrent cancers. However, the clinical relevance of HT is still debated due to a number of challenges, such as tumor specific temperature increase, uneven heating of the target, and the lack of agents that concurrently execute HT in combination with radio- and/or chemotherapy. Here, the application of non-persistent ultrasmall-in-nano gold architectures for synergistic chemo-photothermal treatment of head and neck squamous cell carcinomas (HNSCCs) is presented. The nano-architectures are composed of excretable narrow near-infrared (NIR)-absorbing gold ultrasmall nanoparticles and an endogenously double controlled cisplatin prodrug. The efficiency of the nano-architectures is evaluated on three-dimensional (3D) models of HNSCCs with positive or negative human papillomavirus (HPV) status. The combined treatment causes a more pronounced antitumor action on HPV-positive HNSCCs. Overall, the findings demonstrate the potential clinical relevance of translatable noble metal-based synergistic treatments in tumors management.

Protein Delivery by Peptide-Based Stealth Liposomes: A Biomolecular Insight into Enzyme Replacement Therapy.

Infantile neural ceroid lipofuscinosis (INCL) is a lysosomal storage disorder characterized by mutations in the CLN1 gene that leads to lack of the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1), which causes the progressive death of cortical neurons. Enzyme replacement therapy (ERT) is one of the most promising treatments, but its translation toward a clinical use is hampered by the need to deliver the enzyme to the central nervous system and a more detailed understanding of its capability to restore physiologic conditions at the biochemical and protein level, beyond the simple regulation of enzymatic activity. Targeted nanoparticles can promote protein delivery to the central nervous system and affect biological pathways inside cells. Here, we describe an innovative peptide-based stealth nanoparticle that inhibits serum protein adsorption exploiting transferrin-driven internalization to convey the PPT1 enzyme to transferrin receptor-mediated pathways (endocytosis in this work, or transcytosis, in perspective, in vivo). These enzyme-loaded nanoparticles were able to restore stable levels of enzymatic activity in CLN1 patient's fibroblasts, comparable with the free enzyme, demonstrating that delivery after encapsulation in the nanocarrier does not alter uptake or intracellular trafficking. We also investigate, for the first time, dysregulated pathways of proteome and palmitoylome and their alteration upon enzyme delivery. Our nanoparticles were able of halving palmitoylated protein levels restoring conditions similar to the normal cells. From proteomic analysis, we also highlighted the reduction of the different groups of proteins after treatments with the free or encapsulated enzyme. In conclusion, our system is able to deliver the enzyme to a model of CLN1 disease restoring normal conditions in cells. Investigation of molecular details of pathologic state and enzyme-based correction reveals dysregulated pathways with unprecedented details for CLN1. Finally, we unveil for the first time the dysregulation landscape of palmitoylome and proteome in primary patient-derived fibroblasts and their modifications in response to enzyme administration. These findings will provide a guideline for the validation of future therapeutic strategies based on enzyme replacement therapy or acting at different metabolic levels.

A Cost-Effective Approach for Non-Persistent Gold Nano-Architectures Production.

The effective exploitation of the intriguing theranostic features of noble metal nanoparticles for therapeutic applications is far from being a routine practice due to the persistence issue. In this regard, passion fruit-like nano-architectures (NAs), biodegradable and excretable all-in-one, nature-inspired platforms which jointly combine these characteristics with the appealing optical behaviors of noble metal nanoparticles, can offer a new alternative for theranostic applications. Besides the need for efficacious and innovative systems, the reliable and cost-effective production of nanomaterials is a pivotal subject for their translation to the clinical setting. Here, we demonstrate the production of a new cheaper class of degradable, ultrasmall-in-nano-architectures (dragon fruit NAs, dNAs) using polyethyleneimine (PEI) as a cationic polymer without affecting either their compositions or their physiological behaviors, compared to the previous NAs. In particular, the standardized protocol characterized in this work ensures the preparation of high gold-loading capacity nanoparticles, a peculiar characteristic that, synergically with the interesting properties of PEI, may unlock new possible applications previously precluded to the first version of NAs while reducing the hand-made production cost by three orders of magnitude.

Uranium-free X solution: a new generation contrast agent for biological samples ultrastructure.

Biological samples are mainly composed of elements with a low atomic number which show a relatively low electron scattering power. For Transmission Electron Microscopy analysis, biological samples are generally embedded in resins, which allow thin sectioning of the specimen. Embedding resins are also composed by light atoms, thus the contrast difference between the biological sample and the surrounding resin is minimal. Due to that reason in the last decades, several staining solutions and approaches, performed with heavy metal salts, have been developed with the purpose of enhancing both the intrinsic sample contrast and the differences between the sample and resin. The best staining was achieved with the uranyl acetate (UA) solution, which has been the election method for the study of morphology in biological samples. More recently several alternatives for UA have been proposed to get rid of its radiogenic issues, but to date none of these solutions has achieved efficiencies comparable to UA. In this work, we propose a different staining solution (X Solution or X SOL), characterized by lanthanide polyoxometalates (LnPOMs) as heavy atoms source, which could be used alternatively to UA in negative staining (NS), in en bloc staining, and post sectioning staining (PSS) of biological samples. Furthermore, we show an extensive chemical characterization of the LnPOM species present in the solution and the detailed work for its final formulation, which brought remarkable results, and even better performances than UA.

Targeted Dendrimer-Coated Magnetic Nanoparticles for Selective Delivery of Therapeutics in Living Cells.

Nanoparticles are widely used as theranostic agents for the treatment of various pathologies, including cancer. Among all, dendrimers-based nanoparticles represent a valid approach for drugs delivery, thanks to their controllable size and surface properties. Indeed, dendrimers can be easily loaded with different payloads and functionalized with targeting agents. Moreover, they can be used in combination with other materials such as metal nanoparticles for combinatorial therapies. Here, we present the formulation of an innovative nanostructured hybrid system composed by a metallic core and a dendrimers-based coating that is able to deliver doxorubicin specifically to cancer cells through a targeting agent. Its dual nature allows us to transport nanoparticles to our site of interest through the magnetic field and specifically increase internalization by exploiting the T7 targeting peptide. Our system can release the drug in a controlled pH-dependent way, causing more than 50% of cell death in a pancreatic cancer cell line. Finally, we show how the system was internalized inside cancer cells, highlighting a peculiar disassembly of the nanostructure at the cell surface. Indeed, only the dendrimeric portion is internalized, while the metal core remains outside. Thanks to these features, our nanosystem can be exploited for a multistage magnetic vector.

Endogenously-Activated Ultrasmall-in-Nano Therapeutics: Assessment on 3D Head and Neck Squamous Cell Carcinomas.

Negative or positive HPV-associated Head and Neck Squamous Cell Carcinomas (HNSCCs) are high recurrence neoplasms usually resulting in a poor prognosis, mainly due to metastasis formation. Despite the low overall patient survival rate and the severe side effects, the treatment of choice is still cisplatin-based chemotherapy. Here, we report a straightforward protocol for the production of high throughput 3D models of negative or positive HPV-associated HNSCCs, together with their employment in the therapeutic evaluation of gold ultrasmall-in-nano architectures comprising an endogenously-activatable cisplatin prodrug. Beyond enhancing the biosafety of cisplatin, our approach paves the way for the establishment of synergistic co-therapies for HNSCCs based on excretable noble metals.

Biokinetics and clearance of inhaled gold ultrasmall-in-nano architectures.

Among an organism's entry portals, the respiratory tract is one of the most promising routes for non-invasive administration of therapeutics for local and systemic delivery. On the other hand, it is the subtlest to protect from environmental pollution and microbial occurrences. Here, the biokinetics, distribution, and clearance trends of gold ultrasmall-in-nano architectures administered through a single intranasal application have been quantitatively evaluated. Apart from reaching the lung parenchyma, the (bio)degradable nano-architectures are able to translocate as well to secondary organs and be almost completely excreted within 10 days. These findings further support the clinical relevance of plasmonic nanomaterials for oncology and infectious disease treatment and management. Notably, this investigation also provides crucial information regarding the associated risks as a consequence of the pulmonary delivery of nanoparticles.

Production of 3D Tumor Models of Head and Neck Squamous Cell Carcinomas for Nanotheranostics Assessment.

As a first approach, standard 2D cell culture techniques are usually employed for the screening of drugs and nanomaterials. Despite the easy handling, findings achieved on 2D cultures are often not efficiently translatable to in vivo preclinical investigations. Furthermore, although animal models are pivotal in preclinical studies, more strict directives have been implemented to promote the use of alternative biological systems. In this context, the development and integration into preclinical research workflow of 3D neoplasm models is particularly appealing to promote the advancement and success of therapeutics in clinical trials while reducing the number of in vivo models. Indeed, 3D tumor models bridge several discrepancies between 2D cell culture and in vivo models, among which are morphology, polarity, drug penetration, osmolality, and gene expressions. Here, we comprehensively describe a robust and high-throughput hanging drop protocol for the production of 3D models of both Human Papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas (HNSCCs). We also report the standard cascade assays for their characterization and demonstrate their significance in investigations on these aggressive neoplasms. The employment of relevant 3D cancer models is pivotal to produce more reliable and robust findings in terms of biosafety, theranostic efficacy, and biokinetics as well as to promote further knowledge on HNSCC pathophysiology.

Brain-targeted enzyme-loaded nanoparticles: A breach through the blood-brain barrier for enzyme replacement therapy in Krabbe disease.

Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement.

Biosafety and Biokinetics of Noble Metals: The Impact of Their Chemical Nature.

Effective excretion of nanostructured noble metals is still one of the most challenging bottlenecks for their employment in clinical practice. Besides the persistence issue, the clinical translation of inorganic nanomaterials is also affected by a bewildering lack of investigations regarding their quantitative biokinetics. Here, we have quantitatively correlated the chemical nature of the three most interesting noble metals for biomedical applications to their biosafety and biokinetics in, respectively, zebrafish and murine models. Gold, silver, and platinum ultrasmall-in-nano architectures with comparable size elicit, after intravenous administration, different excretion pathways depending on their intrinsic metallic nature. Understanding the in vivo fate of noble metal nanoparticles is a significant breakthrough to unlock their clinical employment for the establishment of treatments for neoplasms, infectious diseases, and neurological disorders.

Endogenously Triggerable Ultrasmall-in-Nano Architectures: Targeting Assessment on 3D Pancreatic Carcinoma Spheroids.

Several nanomaterials rely on the passive accumulation in the neoplasm target because of enhanced permeability and retention effect. On the other hand, directing nanomaterials to the target by employing the targeting agents may lead to a pivotal improvement in the efficacy of the treatment for a number of cancers. However, targeting moieties often lose their functionality upon injection in the bloodstream, leaving questions on their efficiency. Here, we assessed using a significant in vitro 3D model of pancreatic carcinoma the targeting efficiency of passion fruit-like nanoarchitectures (NAs) incorporated with a peptide that can recognize transferrin directly in the medium, thereby modulating protein solvation. NAs are biodegradable ultrasmall-in-nano platforms that combine the most appealing behaviors of noble metal nanomaterials with organism excretion of the building blocks by the renal pathway. Although the confocal images did not illustrate the significant differences in the targeting efficiency of the peptide-modified NAs, an improved internalization was quantitatively observed by inductively coupled plasma-mass spectrometry analysis. Our findings demonstrate that the peptide conjugation of NAs might be considered to enhance their theranostic potentials for this type of neoplasm.

Cross-Linked Enzyme Aggregates as Versatile Tool for Enzyme Delivery: Application to Polymeric Nanoparticles.

Polymeric nanoparticles (NPs) represent one of the most promising tools in nanomedicine and have been extensively studied for the delivery of water-insoluble drugs. However, the efficient loading of therapeutic enzymes and proteins in polymer-based nanostructures remains an open challenge. Here, we report a synthesis method for a new enzyme delivery system based on cross-linked enzyme aggregates (CLEAs) encapsulation into poly(lactide- co-glycolide) (PLGA) NPs. We tested the encapsulation strategy on four enzymes currently investigated for enzyme replacement therapy: palmitoyl protein thioesterase 1 (PPT1; defective in NCL1 disease), galactosylceramidase (GALC; defective in globoid cell leukodystrophy), alpha glucosidase (aGLU; defective in Pompe disease), and beta glucosidase (bGLU; defective in Gaucher's disease). We demonstrated that our system allows encapsulation of enzymes with excellent activity retention (usually around 60%), thus leading to functional and targeted nanostructures suitable for enzyme delivery. We then demonstrated that CLEA NPs efficiently deliver PPT1 in cultured cells, with almost complete enzyme release occurring in 48 h. Finally, we demonstrated that enzymatic activity is fully recovered in primary NCL1 fibroblasts upon treatment with PPT1 CLEA NPs.

Peptide-Based Stealth Nanoparticles for Targeted and pH-Triggered Delivery.

Stealth agents are extensively investigated as a means by which to prolong nanostructure residence time in the bloodstream by avoiding uptake by the reticuloendothelial system. Unfortunately, commonly used agents such as poly(ethylene glycol) can adversely impact targeting efficiency and promote immune reaction by the host organism. Therefore, there is an increasing interest in developing biocompatible, non-PEGylated organic nanostructures able to perform targeted delivery to increase the efficacy of liposomal technology. Here, a lipopeptide is presented that can be mixed with lipids commonly used in liposomal formulations in percentages ranging from 20% to 60% w/w. The resulting vesicles are thermally and chemically stable. The peptide coating limits serum-protein adsorption even upon prolonged incubation in pure serum in physiological conditions, outperforming PEGylated liposomes. This architecture can be easily modified to allow straightforward derivatization by standard bio-orthogonal conjugation. Upon derivatization with an anti-transferrin receptor aptamer, these vesicles show highly selective cellular internalization with minimal nonspecific uptake and pH-triggered doxorubicin release.

Rational Design of a Transferrin-Binding Peptide Sequence Tailored to Targeted Nanoparticle Internalization.

The transferrin receptor (TfR) is a promising target in cancer therapy owing to its overexpression in most solid tumors and on the blood-brain barrier. Nanostructures chemically derivatized with transferrin are employed in TfR targeting but often lose their functionality upon injection in the bloodstream. As an alternative strategy, we rationally designed a peptide coating able to bind transferrin on suitable pockets not involved in binding to TfR or iron by using an iterative multiscale-modeling approach coupled with quantitative structure-activity and relationship (QSAR) analysis and evolutionary algorithms. We tested that selected sequences have low aspecific protein adsorption and high binding energy toward transferrin, and one of them is efficiently internalized in cells with a transferrin-dependent pathway. Furthermore, it promotes transferrin-mediated endocytosis of gold nanoparticles by modifying their protein corona and promoting oriented adsorption of transferrin. This strategy leads to highly effective nanostructures, potentially useful in diagnostic and therapeutic applications, which exploit (and do not suffer) the protein solvation for achieving a better targeting.