Lipoplexes for effective in vitro delivery of microRNAs to adult human cardiac fibroblasts for perspective direct cardiac cell reprogramming.

Design of nanocarriers for efficient miRNA delivery can significantly improve miRNA-based therapies. Lipoplexes based on helper lipid, dioleoyl phosphatidylethanolamine (DOPE) and cationic lipid [2-(2,3-didodecyloxypropyl)-hydroxyethyl] ammonium bromide (DE) were formulated to efficiently deliver miR-1 or a combination of four microRNAs (miRcombo) to adult human cardiac fibroblasts (AHCFs). Lipoplexes with amino-to-phosphate groups ratio of 3 (N/P 3) showed nanometric hydrodynamic size (372 nm), positive Z-potential (40 mV) and high stability under storage conditions. Compared to commercial DharmaFECT1 (DF), DE-DOPE/miRNA lipoplexes showed superior miRNA loading efficiency (99 % vs. 64 %), and faster miRNA release (99 % vs. 82 % at 48 h). DE-DOPE/miR-1 lipoplexes showed superior viability (80-100 % vs. 50 %) in AHCFs, a 2-fold higher miR-1 expression and Twinfilin-1 (TWF-1) mRNA downregulation. DE-DOPE/miRcombo lipoplexes significantly enhanced AHCFs reprogramming into induced cardiomyocytes (iCMs), as shown by increased expression of CM markers compared to DF/miRcombo.

L-Carnitine Functionalization to Increase Skeletal Muscle Tropism of PLGA Nanoparticles.

Muscular dystrophies are a group of rare genetic pathologies, encompassing a variety of clinical phenotypes and mechanisms of disease. Several compounds have been proposed to treat compromised muscles, but it is known that pharmacokinetics and pharmacodynamics problems could occur. To solve these issues, it has been suggested that nanocarriers could be used to allow controlled and targeted drug release. Therefore, the aim of this study was to prepare actively targeted poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) for the treatment of muscular pathologies. By taking advantage of the high affinity for carnitine of skeletal muscle cells due to the expression of Na+-coupled carnitine transporter (OCTN), NPs have been actively targeted via association to an amphiphilic derivative of L-carnitine. Furthermore, pentamidine, an old drug repurposed for its positive effects on myotonic dystrophy type I, was incorporated into NPs. We obtained monodispersed targeted NPs, with a mean diameter of about 100 nm and a negative zeta potential. To assess the targeting ability of the NPs, cell uptake studies were performed on C2C12 myoblasts and myotubes using confocal and transmission electron microscopy. The results showed an increased uptake of carnitine-functionalized NPs compared to nontargeted carriers in myotubes, which was probably due to the interaction with OCTN receptors occurring in large amounts in these differentiated muscle cells.

Hyaluronated and PEGylated Liposomes as a Potential Drug-Delivery Strategy to Specifically Target Liver Cancer and Inflammatory Cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and is characterized by poor clinical outcomes, with the majority of patients not being eligible for curative therapy and treatments only being applicable for early-stage tumors. CD44 is a receptor for hyaluronic acid (HA) and is involved in HCC progression. The aim of this work is to propose HA- and PEGylated-liposomes as promising approaches for the treatment of HCC. It has been found, in this work, that CD44 transcripts are up-regulated in HCC patients, as well as in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Cell culture experiments indicate that HA-liposomes are more rapidly and significantly internalized by Huh7 cells that over-express CD44, compared with HepG2 cells that express low levels of the receptor, in which the uptake seems due to endocytic events. By contrast, human and murine macrophage cell lines (THP-1, RAW264.7) show improved and rapid uptake of PEG-modified liposomes without the involvement of the CD44. Moreover, the internalization of PEG-modified liposomes seems to induce polarization of THP1 towards the M1 phenotype. In conclusion, data reported in this study indicate that this strategy can be proposed as an alternative for drug delivery and one that dually and specifically targets liver cancer cells and infiltrating tumor macrophages in order to counteract two crucial aspect of HCC progression.

Investigation of squalene-doxorubicin distribution and interactions within single cancer cell using Raman microspectroscopy.

Intracellular distribution of doxorubicin (DOX) and its squalenoylated (SQ-DOX) nanoparticles (NPs) form in murine lung carcinoma M109 and human breast carcinoma MDA-MB-231 cells was investigated by Raman microspectroscopy. Pharmacological data showed that DOX induced higher cytotoxic effect than SQ-DOX NPs. Raman data were obtained using single-point measurements and imaging on the whole cell areas. These data showed that after DOX treatment at 1 µM, the spectral features of DOX were not detected in the M109 cell cytoplasm and nucleus. However, the intracellular distribution of SQ-DOX NPs was higher than DOX in the same conditions. In addition, SQ-DOX NPs were localized into both cell cytoplasm and nucleus. After 5 µM treatment, Raman bands of DOX at 1211 and 1241 cm-1 were detected in the nucleus. Moreover, the intensity ratio of these bands decreased, indicating DOX intercalation into DNA. However, after treatment with SQ-DOX NPs, the intensity of these Raman bands increased. Interestingly, with SQ-DOX NPs, the intensity of 1210/1241 cm-1 ratio was higher suggesting a lower fraction of intercalated DOX in DNA and higher amount of non-hydrolyzed SQ-DOX. Raman imaging data confirm this subcellular localization of these drugs in both M109 and MDA-MB-231 cells. These finding brings new insights to the cellular characterization of anticancer drugs at the molecular level, particularly in the field of nanomedicine.

New Formulation for the Delivery of Oligonucleotides Using "Clickable" siRNA-Polyisoprenoid-Conjugated Nanoparticles: Application to Cancers Harboring Fusion Oncogenes.

The aim of the present study is to take advantage of the unique property of polyisoprenoid chains to adopt a compact molecular conformation and to use these natural and biocompatible lipids as nanocarriers of drugs to deliver siRNA. A new chemical strategy is applied here to conjugate squalene (SQ) and solanesol (SOLA) to siRNA consisting of an activated variant of the azide-alkyne Huisgen cycloaddition also known as copper-free (Cu-free) click chemistry. We conjugated siRNA against TMPRSS2-ERG, a fusion oncogene found in more than 50% of prostate cancers to SQ or SOLA. First, several parameters such as molar ratio, solvents, temperature, incubation time, and the annealing schedule between both siRNA strands were investigated to bioconjugate the SQ or SOLA via Cu-free click chemistry. The best parameters of the new bioconjugation approach allowed us to (i) increase the synthesis yield up to 95%, (ii) avoid the formation of byproducts during the synthesis, and (iii) improve the reproducibility of the bioconjugation. Then, the biological activity of the resulting nanoparticles was assessed. In vitro, all four formulations were able to decrease the corresponding oncogene and oncoprotein expression. In vivo, only two of the four nanoformulations showed anti-neoplastic activity that seems to be tightly related to their dissimilar biodistribution behavior. In conclusion, we performed a new approach easily transposable for pharmaceutical development to synthesize siRNA-SQ and siRNA-SOLA and to obtain efficient siRNA-nanoparticles. The robustness of the process could be extended to several other polyterpenes and likely applied to other siRNA targeting genes whose overexpression results in the development of cancers or other genetic diseases.

Nanoparticles obtained by confined impinging jet mixer: poly(lactide-co-glycolide) vs. Poly-ε-caprolactone.

This paper is focused on the production and characterization of polymeric nanoparticles obtained by nanoprecipitation. The method consisted of using a confined impinging jet mixer (CIJM), circumventing high-energy equipment. Differences between the use of poly-ε-caprolactone (PCL) and poly(lactide-co-glycolide) (PLGA) as concerns particle mean size, zeta potential, and broad-spectrum antibiotic florfenicol entrapment were investigated. Other analyzed variables were polymer concentration, solvent, and anti-solvent flow rates, and antibiotic initial concentration. To our knowledge, no data were found related to PLGA and PCL nanoparticles comparison using CIJM. Also, florfenicol encapsulation within PCL or PLGA nanoparticles by nanoprecipitation has not been reported yet. The complexity of the nanoprecipitation phenomena has been confirmed, with many relevant variables involved in particles formation. PLGA resulted in smaller and more stable nanoparticles with higher entrapping of florfenicol than PCL.

A unique squalenoylated and nonpegylated doxorubicin nanomedicine with systemic long-circulating properties and anticancer activity.

We identified that the chemical linkage of the anticancer drug doxorubicin onto squalene, a natural lipid precursor of the cholesterol's biosynthesis, led to the formation of squalenoyl doxorubicin (SQ-Dox) nanoassemblies of 130-nm mean diameter, with an original "loop-train" structure. This unique nanomedicine demonstrates: (i) high drug payload, (ii) decreased toxicity of the coupled anticancer compound, (iii) improved therapeutic response, (iv) use of biocompatible transporter material, and (v) ease of preparation, all criteria that are not combined in the currently available nanodrugs. Cell culture viability tests and apoptosis assays showed that SQ-Dox nanoassemblies displayed comparable antiproliferative and cytotoxic effects than the native doxorubicin because of the high activity of apoptotic mediators, such as caspase-3 and poly(ADP-ribose) polymerase. In vivo experiments have shown that the SQ-Dox nanomedicine dramatically improved the anticancer efficacy, compared with free doxorubicin. Particularly, the M109 lung tumors that did not respond to doxorubicin treatment were found inhibited by 90% when treated with SQ-Dox nanoassemblies. SQ-Dox nanoassembly-treated MiaPaCa-2 pancreatic tumor xenografts in mice decreased by 95% compared with the tumors in the saline-treated mice, which was significantly higher than the 29% reduction achieved by native doxorubicin. Concerning toxicity, SQ-Dox nanoassemblies showed a fivefold higher maximum-tolerated dose than the free drug, and moreover, the cardiotoxicity study has evidenced that SQ-Dox nanoassemblies did not cause any myocardial lesions, such as those induced by the free doxorubicin treatment. Taken together, these findings demonstrate that SQ-Dox nanoassemblies make tumor cells more sensitive to doxorubicin and reduce the cardiac toxicity, thus providing a remarkable improvement in the drug's therapeutic index.

Peptide conjugation: before or after nanoparticle formation?

We report herein a detailed study concerning the impact of different bioconjugation and nanoformulation strategies on the in vitro targeting ability of peptide-decorated squalenoyl gemcitabine (SQdFdC) nanoparticles (NPs). NPs have been functionalized with the CKAAKN peptide, previously identified as an efficient homing device within the pancreatic pathological microenvironment. Two approaches have been followed: (i) either the CKAAKN peptide was directly conjugated at the surface of preformed SQdFdC nanoparticles (conjugation after NP formation) or (ii) it was first reacted with a maleimide squalenoyl derivative before the resulting bioconjugate was co-nanoprecipitated with SQdFdC to form the peptide-decorated NPs (conjugation before NP formation). NPs were characterized with respect to mean diameter, zeta potential, and stability over time. Then, their specific interaction with the sFRP-4 protein was evaluated by surface plasmon resonance. Although both synthetic strategies allowed us to formulate NPs able to interact with the corresponding receptor, enhanced target binding and better specific avidity were observed with CKAAKN-NPs functionalized before NP formation. These NPs displayed the highest cell uptake and cytotoxicity in an in vitro model of human MIA Paca-2 pancreatic cancer cells.

Hyaluronic acid conjugates as vectors for the active targeting of drugs, genes and nanocomposites in cancer treatment.

Hyaluronic acid (HA) is a naturally-occurring glycosaminoglycan and a major component of the extracellular matrix. Low levels of the hyaluronic acid receptor CD44 are found on the surface of epithelial, hematopoietic, and neuronal cells; it is overexpressed in many cancer cells, and in particular in tumor-initiating cells. HA has recently attracted considerable interest in the field of developing drug delivery systems, having been used, as such or encapsulated in different types of nanoassembly, as ligand to prepare nano-platforms for actively targeting drugs, genes, and diagnostic agents. This review describes recent progress made with the several chemical strategies adopted to synthesize conjugates and prepare novel delivery systems with improved behaviors.

Targeting pentamidine towards CD44-overexpressing cells using hyaluronated lipid-polymer hybrid nanoparticles.

Biodegradable nanocarriers possess enormous potential for use as drug delivery systems that can accomplish controlled and targeted drug release, and a wide range of nanosystems have been reported for the treatment and/or diagnosis of various diseases and disorders. Of the various nanocarriers currently available, liposomes and polymer nanoparticles have been extensively studied and some formulations have already reached the market. However, a combination of properties to create a single hybrid system can give these carriers significant advantages, such as improvement in encapsulation efficacy, higher stability, and active targeting towards specific cells or tissues, over lipid or polymer-based platforms. To this aim, this work presents the formulation of poly(lactic-co-glycolic) acid (PLGA) nanoparticles in the presence of a hyaluronic acid (HA)-phospholipid conjugate (HA-DPPE), which was used to anchor HA onto the nanoparticle surface and therefore create an actively targeted hybrid nanosystem. Furthermore, ionic interactions have been proposed for drug encapsulation, leading us to select the free base form of pentamidine (PTM-B) as the model drug. We herein report the preparation of hybrid nanocarriers that were loaded via ion-pairing between the negatively charged PLGA and HA and the positively charged PTM-B, demonstrating an improved loading capacity compared to PLGA-based nanoparticles. The nanocarriers displayed a size of below 150 nm, a negative zeta potential of -35 mV, a core-shell internal arrangement and high encapsulation efficiency (90%). Finally, the ability to be taken up and exert preferential and receptor-mediated cytotoxicity on cancer cells that overexpress the HA specific receptor (CD44) has been evaluated. Competition assays supported the hypothesis that PLGA/HA-DPPE nanoparticles deliver their cargo within cells in a CD44-dependent manner.

Designing functionalized nanodiamonds with hyaluronic acid-phospholipid conjugates for enhanced cancer cell targeting and fluorescence imaging capabilities.

Nanomedicine aims to develop smart approaches for treating cancer and other diseases to improve patient survival and quality of life. Novel nanoparticles as nanodiamonds (NDs) represent promising candidates to overcome current limitations. In this study, NDs were functionalized with a 200 kDa hyaluronic acid-phospholipid conjugate (HA/DMPE), enhancing the stability of the nanoparticles in water-based solutions and selectivity for cancer cells overexpressing specific HA cluster determinant 44 (CD44) receptors. These nanoparticles were characterized by diffuse reflectance Fourier-transform infrared spectroscopy, Raman spectroscopy, and photoluminescence spectroscopy, confirming the efficacy of the functionalization process. Scanning electron microscopy was employed to evaluate the size distribution of the dry particles, while dynamic light scattering and zeta potential measurements were utilized to evaluate ND behavior in a water-based medium. Furthermore, the ND biocompatibility and uptake mediated by CD44 receptors in three different models of human adenocarcinoma cells were assessed by performing cytofluorimetric assay and confocal microscopy. HA-functionalized nanodiamonds demonstrated the advantage of active targeting in the presence of cancer cells expressing CD44 on the surface, suggesting higher drug delivery to tumors over non-tumor tissues. Even CD44-poorly expressing cancers could be targeted by the NDs, thanks to their good passive diffusion within cancer cells.

Rational design for multifunctional non-liposomal lipid-based nanocarriers for cancer management: theory to practice.

Nanomedicines have gained more and more attention in cancer therapy thanks to their ability to enhance the tumour accumulation and the intracellular uptake of drugs while reducing their inactivation and toxicity. In parallel, nanocarriers have been successfully employed as diagnostic tools increasing imaging resolution holding great promises both in preclinical research and in clinical settings. Lipid-based nanocarriers are a class of biocompatible and biodegradable vehicles that provide advanced delivery of therapeutic and imaging agents, improving pharmacokinetic profile and safety. One of most promising engineering challenges is the design of innovative and versatile multifunctional targeted nanotechnologies for cancer treatment and diagnosis. This review aims to highlight rational approaches to design multifunctional non liposomal lipid-based nanocarriers providing an update of literature in this field.

An ex vivo experimental system to track fluorescent nanoparticles inside skeletal muscle.

The development of novel nanoconstructs for biomedical applications requires the assessment of their biodistribution, metabolism and clearance in single cells, organs and entire organisms in a living environment. To reduce the number of in vivo experiments performed and to refine the methods used, in accordance with the 3Rs principle, this work proposes an ex vivo experimental system to monitor, using fluorescence microscopy, the distribution of nanoparticles in explanted murine skeletal muscle maintained in a bioreactor that can preserve the structural and functional features of the organ for long periods of time. Fluorescently-labelled liposomes and poly(lactide-co-glycolide) (PLGA)-based nanoparticles were injected into the intact soleus muscle (in the distal region close to the tendon) immediately after explants, and their distribution was analysed at increasing incubation times in cross cryosections from the proximal region of the belly. Both nanocarriers were clearly recognized in the muscle and were found to enter and migrate inside the myofibres, whereas their migration in the connective tissue seemed to be limited. In addition, some fluorescent signals were observed inside the macrophages, demonstrating the physiological clearance of the nanocarriers that did not enter the myofibres. Our ex vivo system therefore provides more information than previous in vitro experiments on cultured muscle cells, highlighting the need for the appropriate functionalization of nanocarriers if myofibre targeting is to be improved.

Freeze Drying of Polymer Nanoparticles and Liposomes Exploiting Different Saccharide-Based Approaches.

Biodegradable nanocarriers represent promising tools for controlled drug delivery. However, one major drawback related to their use is the long-term stability, which is largely influenced by the presence of water in the formulations, so to solve this problem, freeze-drying with cryoprotectants has been proposed. In the present study, the influence of the freeze-drying procedure on the storage stability of poly(lactide-co-glycolide) (PLGA) nanoparticles and liposomes was evaluated. In particular, conventional cryoprotectants were added to PLGA nanoparticle and liposome formulations in various conditions. Additionally, hyaluronic acid (HA), known for its ability to target the CD44 receptor, was assessed as a cryoprotective excipient: it was added to the nanocarriers as either a free molecule or conjugated to a phospholipid to increase the interaction with the polymer or lipid matrix while exposing HA on the nanocarrier surface. The formulations were resuspended and characterized for size, polydispersity index, zeta potential and morphology. It was demonstrated that only the highest percentages of cryoprotectants allowed the resuspension of stable nanocarriers. Moreover, unlike free HA, HA-phospholipid conjugates were able to maintain the particle mean size after the reconstitution of lyophilized nanoparticles and liposomes. This study paves the way for the use of HA-phospholipids to achieve, at the same time, nanocarrier cryoprotection and active targeting.

Nanotechnological approaches for pentamidine delivery.

Pentamidine (PTM), which is a diamine that is widely known for its antimicrobial activity, is a very interesting drug whose mechanism of action is not fully understood. In recent years, PTM has been proposed as a novel potential drug candidate for the treatment of mental illnesses, myotonic dystrophy, diabetes, and tumors. Nevertheless, the systemic administration of PTM causes severe side effects, especially nephrotoxicity. In order to efficiently deliver PTM and reduce its side effects, several nanosystems that take advantage of the chemical characteristics of PTM, such as the presence of two positively charged amidine groups at physiological pH, have been proposed as useful delivery tools. Polymeric, lipidic, inorganic, and other types of nanocarriers have been reported in the literature for PTM delivery, and they are all in different development phases. The available approaches for the design of PTM nanoparticulate delivery systems are reported in this review, with a particular emphasis on formulation strategies and in vitro/in vivo applications. Furthermore, a critical view of the future developments of nanomedicine for PTM applications, based on recent repurposing studies, is provided. Created with BioRender.com.

Interaction of self-assembled squalenoyl gemcitabine nanoparticles with phospholipid-cholesterol monolayers mimicking a biomembrane.

Gemcitabine (dFdC or Gem) is a water-soluble cytotoxic drug, with poor cellular uptake in the absence of a nucleoside transporter. To improve its diffusion through membranes, it was modified by grafting of a squalenoyl moiety. In water, this derivative is able to form stable and monodispersed nanoparticles made of inverse hexagonal phases. The formation and interfacial properties of the squalenoyl gemcitabine (SQ-Gem) nanoparticles, and their ability to interact with phospholipid and cholesterol monolayers modeling a biomembrane, was assessed from surface tension measurements and Brewster angle microscopy. To get a better insight into the mechanisms of SQ-Gem interaction with the various lipids, the interfacial behavior of SQ-Gem and squalene was also studied by surface pressure and surface potential measurements, in the absence and in the presence of phospholipids and cholesterol. The results showed that SQ-Gem nanoparticles adsorbed at the free air/water interface and disrupted to form a monolayer. SQ-Gem molecules released from the adsorbed nanoparticles were also able to penetrate into condensed phospholipid-cholesterol mixed monolayers. The kinetics of this penetration was apparently controlled by intermolecular interactions between the drug and the adsorbed lipids. Whereas distearoylphosphatidylcholine (DSPC) hindered SQ-Gem penetration, cholesterol favored it, which could have important implications in the therapeutic field since cholesterol targeting could alter lipid raft composition and cancer cell survival.

Cannabinoid Formulations and Delivery Systems: Current and Future Options to Treat Pain.

The field of Cannabis sativa L. research for medical purposes has been rapidly advancing in recent decades and a growing body of evidence suggests that phytocannabinoids are beneficial for a range of conditions. At the same time impressing development has been observed for formulations and delivery systems expanding the potential use of cannabinoids as an effective medical therapy. The objective of this review is to present the most recent results from pharmaceutical companies and research groups investigating methods to improve cannabinoid bioavailability and to clearly establish its therapeutic efficacy, dose ranges, safety and also improve the patient compliance. Particular focus is the application of cannabinoids in pain treatment, describing the principal cannabinoids employed, the most promising delivery systems for each administration routes and updating the clinical evaluations. To offer the reader a wider view, this review discusses the formulation starting from galenic preparation up to nanotechnology approaches, showing advantages, limits, requirements needed. Furthermore, the most recent clinical data and meta-analysis for cannabinoids used in different pain management are summarized, evaluating their real effectiveness, in order also to spare opioids and improve patients' quality of life. Promising evidence for pain treatments and for other important pathologies are also reviewed as likely future directions for cannabinoids formulations.

Developing Actively Targeted Nanoparticles to Fight Cancer: Focus on Italian Research.

Active targeting is a valuable and promising approach with which to enhance the therapeutic efficacy of nanodelivery systems, and the development of tumor-targeted nanoparticles has therefore attracted much research attention. In this field, the research carried out in Italian Pharmaceutical Technology academic groups has been focused on the development of actively targeted nanosystems using a multidisciplinary approach. To highlight these efforts, this review reports a thorough description of the last 10 years of Italian research results on the development of actively targeted nanoparticles to direct drugs towards different receptors that are overexpressed on cancer cells or in the tumor microenvironment. In particular, the review discusses polymeric nanocarriers, liposomes, lipoplexes, niosomes, solid lipid nanoparticles, squalene nanoassemblies and nanobubbles. For each nanocarrier, the main ligands, conjugation strategies and target receptors are described. The literature indicates that polymeric nanoparticles and liposomes stand out as key tools for improving specific drug delivery to the site of action. In addition, solid lipid nanoparticles, squalene nanoparticles and nanobubbles have also been successfully proposed. Taken together, these strategies all offer many platforms for the design of nanocarriers that are suitable for future clinical translation.

Nanomedicine for Gene Delivery and Drug Repurposing in the Treatment of Muscular Dystrophies.

Muscular Dystrophies (MDs) are a group of rare inherited genetic muscular pathologies encompassing a variety of clinical phenotypes, gene mutations and mechanisms of disease. MDs undergo progressive skeletal muscle degeneration causing severe health problems that lead to poor life quality, disability and premature death. There are no available therapies to counteract the causes of these diseases and conventional treatments are administered only to mitigate symptoms. Recent understanding on the pathogenetic mechanisms allowed the development of novel therapeutic strategies based on gene therapy, genome editing CRISPR/Cas9 and drug repurposing approaches. Despite the therapeutic potential of these treatments, once the actives are administered, their instability, susceptibility to degradation and toxicity limit their applications. In this frame, the design of delivery strategies based on nanomedicines holds great promise for MD treatments. This review focuses on nanomedicine approaches able to encapsulate therapeutic agents such as small chemical molecules and oligonucleotides to target the most common MDs such as Duchenne Muscular Dystrophy and the Myotonic Dystrophies. The challenge related to in vitro and in vivo testing of nanosystems in appropriate animal models is also addressed. Finally, the most promising nanomedicine-based strategies are highlighted and a critical view in future developments of nanomedicine for neuromuscular diseases is provided.

Novel nanoassemblies composed of squalenoyl-paclitaxel derivatives: synthesis, characterization, and biological evaluation.

Using the anticancer compound paclitaxel as a model drug, this study investigates the potential of the squalenoylation technology (i.e., bioconjugation with the natural lipid squalene) in addressing the drug ability and delivery issues of poorly soluble therapeutic agents. In this view, a variety of novel squalene-based prodrugs of the anticancer compound paclitaxel were synthesized, which produced nanoparticles in water. These prodrugs were obtained by covalent coupling of the paclitaxel 2'-hydroxyl group as direct ester, as well as with a succinate or a diglycolate ester as cleavable linker to the 1,1',2-tris-norsqualenoic acid. The hydrophilicity of these paclitaxel bioconjugates was increased by placing poly(ethylene glycol) chains of different lengths between paclitaxel and the squalenoyl moiety. All these prodrugs self-assembled into nanosized aggregates in aqueous solution as characterized by dynamic light scattering, atomic force microscopy, and transmission electron microscopy. The critical aggregation concentration was very low, ranging from 0.09 to 0.4 mg/L. Zeta potential measurements revealed that all squalenoyl-paclitaxel nanoassemblies (NA) held a global negative charge and appeared stable in water for several weeks as determined by particle size measurement. The release of paclitaxel from NA was evaluated in different conditions and in the presence of serum and depended on the nature of the linker used. Preliminary biological assessment showed that these squalenoyl-paclitaxel NA induced the formation of microtubule bundles in HT-29 and KB-31 cells, and additionally displayed notable cytotoxicity on a lung tumor cell line. Furthermore, the cytotoxic activity of these different prodrugs correlated closely with the observed linker stability. Overall, the squalenoylation nanotechnology opens up interesting perspectives for the development of injectable prodrugs of poorly soluble therapeutic compounds by addressing the associated physicochemical and biopharmaceutical challenges.