Unlocking the Mitochondria for Nanomedicine-based Treatments: Overcoming Biological Barriers, Improving Designs, and Selecting Verification Techniques.

Enhanced targeting approaches will support the treatment of diseases associated with dysfunctional mitochondria, which play critical roles in energy generation and cell survival. Obstacles to mitochondria-specific targeting include the presence of distinct biological barriers and the need to pass through (or avoid) various cell internalization mechanisms. A range of studies have reported the design of mitochondrially-targeted nanomedicines that navigate the complex routes required to influence mitochondrial function; nonetheless, a significant journey lies ahead before mitochondrially-targeted nanomedicines become suitable for clinical use. Moving swiftly forward will require safety studies, in vivo assays confirming effectiveness, and methodologies to validate mitochondria-targeted nanomedicines' subcellular location/activity. From a nanomedicine standpoint, we describe the biological routes involved (from administration to arrival within the mitochondria), the features influencing rational design, and the techniques used to identify/validate successful targeting. Overall, rationally-designed mitochondria-targeted-based nanomedicines hold great promise for precise subcellular therapeutic delivery.

Critical Design Strategies Supporting Optimized Drug Release from Polymer-Drug Conjugates.

The importance of an adequate linking moiety design that allows controlled drug(s) release at the desired site of action is extensively studied for polymer-drug conjugates (PDCs). Redox-responsive self-immolative linkers bearing disulfide moieties (SS-SIL) represent a powerful strategy for intracellular drug delivery; however, the influence of drug structural features and linker-associated spacers on release kinetics remains relatively unexplored. The influence of drug/spacer chemical structure and the chemical group available for conjugation on drug release and the biological effect of resultant PDCs is evaluated. A "design of experiments" tool is implemented to develop a liquid chromatography-mass spectrometry method to perform the comprehensive characterization required for this systematic study. The obtained fit-for-purpose analytical protocol enables the quantification of low drug concentrations in drug release studies and the elucidation of metabolite presence. and provides the first data that clarifies how drug structural features influence the drug release from SS-SIL and demonstrates the non-universal nature of the SS-SIL. The importance of rigorous linker characterization in understanding structure-function correlations between linkers, drug chemical functionalities, and in vitro release kinetics from a rationally-designed polymer-drug nanoconjugate, a critical strategic crafting methodology that should remain under consideration when using a reductive environment as an endogenous drug release trigger.

Rational design of poly-L-glutamic acid-palbociclib conjugates for pediatric glioma treatment.

Brain tumors represent the second most common cause of pediatric cancer death, with malignant gliomas accounting for ∼75% of pediatric deaths. Palbociclib, a selective cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, has shown promise in phase I clinical trials of pediatric patients with progressive/refractory brain tumors using the oral administration route; however, pharmacokinetic limitations and toxicity issues remain. We synthesized a family of well-defined linear and star-shaped polyglutamate (PGA)-palbociclib conjugates using redox-sensitive self-immolative linkers to overcome limitations associated with free palbociclib. Exhaustive characterization of this conjugate family provided evidence for a transition towards the formation of more organized conformational structures upon increased drug loading. We evaluated the activity of conjugates in patient-derived glioblastoma and diffuse intrinsic pontine glioma cells, which display differing reducing environments due to differential glutathione expression levels. We discovered that microenvironmental parameters and the identified conformational changes determined palbociclib release kinetics and therapeutic output; furthermore, we identified a star-shaped PGA-palbociclib conjugate with low drug loading as an optimal therapeutic approach in diffuse intrinsic pontine glioma cells.

Current hurdles to the translation of nanomedicines from bench to the clinic.

The field of nanomedicine has significantly influenced research areas such as drug delivery, diagnostics, theranostics, and regenerative medicine; however, the further development of this field will face significant challenges at the regulatory level if related guidance remains unclear and unconsolidated. This review describes those features and pathways crucial to the clinical translation of nanomedicine and highlights considerations for early-stage product development. These include identifying those critical quality attributes of the drug product essential for activity and safety, appropriate analytical methods (physical, chemical, biological) for characterization, important process parameters, and adequate pre-clinical models. Additional concerns include the evaluation of batch-to-batch consistency and considerations regarding scaling up that will ensure a successful reproducible manufacturing process. Furthermore, we advise close collaboration with regulatory agencies from the early stages of development to assure an aligned position to accelerate the development of future nanomedicines.

Sphingomyelin nanosystems loaded with uroguanylin and etoposide for treating metastatic colorectal cancer.

Colorectal cancer is the third most frequently diagnosed cancer malignancy and the second leading cause of cancer-related deaths worldwide. Therefore, it is of utmost importance to provide new therapeutic options that can improve survival. Sphingomyelin nanosystems (SNs) are a promising type of nanocarriers with potential for association of different types of drugs and, thus, for the development of combination treatments. In this work we propose the chemical modification of uroguanylin, a natural ligand for the Guanylyl Cyclase (GCC) receptor, expressed in metastatic colorectal cancer tumors, to favour its anchoring to SNs (UroGm-SNs). The anti-cancer drug etoposide (Etp) was additionally encapsulated for the development of a combination strategy (UroGm-Etp-SNs). Results from in vitro studies showed that UroGm-Etp-SNs can interact with colorectal cancer cells that express the GCC receptor and mediate an antiproliferative response, which is more remarkable for the drugs in combination. The potential of UroGm-Etp-SNs to treat metastatic colorectal cancer cells was complemented with an in vivo experiment in a xenograft mice model.

Therapeutic potential of polypeptide-based conjugates: Rational design and analytical tools that can boost clinical translation.

The clinical success of polypeptides as polymeric drugs, covered by the umbrella term "polymer therapeutics," combined with related scientific and technological breakthroughs, explain their exponential growth in the development of polypeptide-drug conjugates as therapeutic agents. A deeper understanding of the biology at relevant pathological sites and the critical biological barriers faced, combined with advances regarding controlled polymerization techniques, material bioresponsiveness, analytical methods, and scale up-manufacture processes, have fostered the development of these nature-mimicking entities. Now, engineered polypeptides have the potential to combat current challenges in the advanced drug delivery field. In this review, we will discuss examples of polypeptide-drug conjugates as single or combination therapies in both preclinical and clinical studies as therapeutics and molecular imaging tools. Importantly, we will critically discuss relevant examples to highlight those parameters relevant to their rational design, such as linking chemistry, the analytical strategies employed, and their physicochemical and biological characterization, that will foster their rapid clinical translation.

Envisioning the future of polymer therapeutics for brain disorders.

The growing incidence of brain-related pathologies and the problems that undermine the development of efficient and effective treatments have prompted both researchers and the pharmaceutical industry to search for novel therapeutic alternatives. Polymer therapeutics (PT) display properties well suited to the treatment of neuro-related disorders, which help to overcome the many hidden obstacles on the journey to the central nervous system (CNS). The inherent features of PT, derived from drug(s) conjugation, in parallel with the progress in synthesis and analytical methods, the increasing knowledge in molecular basis of diseases, and collected clinical data through the last four decades, have driven the translation from "bench to bedside" for various biomedical applications. However, since the approval of Gliadel® wafers, little progress has been made in the CNS field, even though brain targeting represents an ever-growing challenge. A thorough assessment of the steps required for successful brain delivery via different administration routes and the consideration of the disease-specific hallmarks are essential to progress in the field. Within this review, we hope to summarize the latest developments, successes, and failures and discuss considerations on designs and strategies for PT in the treatment of CNS disorders. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Lipid-based nanocarriers for oral peptide delivery.

This article is aimed to overview the lipid-based nanostructures designed so far for the oral administration of peptides and proteins, and to analyze the influence of their composition and physicochemical (particle size, zeta potential) and pharmaceutical (drug loading and release) properties, on their interaction with the gastro-intestinal environment, and the subsequent PK/PD profile of the associated drugs. The ultimate goal has been to highlight and comparatively analyze the key factors that may be determinant of the success of these nanocarriers for oral peptide delivery. The article ends with some prospects on the challenges to be addressed for the intended commercial success of these delivery vehicles.

Polymer-doxycycline conjugates as fibril disrupters: an approach towards the treatment of a rare amyloidotic disease.

The term amyloidosis describes neurological diseases where an abnormal protein is misfolded and accumulated as deposits in organs and tissues, known as amyloid, disrupting their normal function. In the most common familial amyloid polyneuropathy (FAP), transthyretin (TTR) displays this role primarily affecting the peripheral nervous system (PNS). Advanced stages of this inherited rare amyloidosis, present as fibril deposits that are responsible for disease progression. In order to stop disease progression, herein we designed an efficient family of nanoconjugates as fibril disrupters. These polymer conjugates are based on doxycycline (doxy), already in phase II trials for Alzheimer's disease, covalently linked to poly-l-glutamic acid (PGA). The conjugates were rationally designed, looking at drug loading and drug release rate by adequate linker design, always considering the physiological conditions at the molecular target site. Conjugation of doxycycline exhibited greater potential towards TTR fibril disaggregation in vitro compared to the parent drug. Exhaustive physico-chemical evaluation of these polymer-drug conjugates concluded that drug release was unnecessary for activity, highlighting the importance of an appropriate linker. Furthermore, biodistribution studies through optical imaging (OI) and the use of radiolabelled polymer-drug conjugates demonstrated conjugate safety profile and renal clearance route of the selected PGA-doxy candidate, settling the adequacy of our conjugate for future in vivo evaluation. Furthermore, preliminary studies in an FAP in vivo model at early stages of disease development showed non-organ toxicity evidences. This nanosized-system raises a promising treatment for advanced stages of this rare amyloidotic disease, and also presents a starting point for possible application within other amyloidosis-related diseases, such as Alzheimer's disease.

Targeting a rare amyloidotic disease through rationally designed polymer conjugates.

Saraiva et al. discovered in 2006 a RAGE-based peptide sequence capable of preventing transthyretin (TTR) aggregate-induced cytotoxicity, hallmark of initial stages of an inherited rare amyloidosis known as Familial Amyloidotic Polyneuropathy (FAP). To allow clinical progression of this peptidic sequence as FAP treatment, a family of polymer conjugates has been designed, synthesised and fully characterised. This approach fulfils the strategies defined in the Polymer Therapeutics area as an exhaustive physico-chemical characterisation fitting activity output towards a novel molecular target that is described here. RAGE peptide acts extracellularly, therefore, no intracellular drug delivery was necessary. PEG was selected as carrier and polymer-drug linker optimisation was then carried out by means of biodegradable (disulphide) and non-biodegradable (amide) covalent bonds. Conjugate size in solution, stability under in vitro and in vivo scenarios and TTR binding affinity through surface plasmon resonance (SPR) was also performed with all synthesised conjugates. In their in vitro evaluation by monitoring the activation of caspase-3 in Schwann cells, peptide derivatives demonstrated retention of peptide activity reducing TTR aggregates (TTRagg) cytotoxicity upon conjugation and a greater plasma stability than the parent free peptide. The results also confirmed that a more stable polymer-peptide linker (amide) is required to secure therapeutic efficiency.