Lipo-polymeric nano-complexes for dermal delivery of a model protein.

Delivering macromolecules across the skin poses challenges due to the barrier properties of stratum corneum. Different strategies have been reported to cross this barrier, such as chemical penetration enhancers and physical methods like microneedles, sonophoresis, electroporation, laser ablation, etc. Herein, we explored a cationic lipo-polymeric nanocarrier to deliver a model protein across the skin. A cationic amphiphilic lipo-polymer was used to prepare blank nanoplexes, which were subsequently complexed with anionic fluorescein-tagged bovine serum albumin (FITC-BSA). Blank nanoplexes and FITC-BSA complexed nanoplexes showed sizes of 93.72 ± 5.8 (PDI-0.250) and 145.9 ± 3.2 nm (PDI-0.258), respectively, and zeta potentials of 25.6 ± 7.0 mV and 9.17 ± 1.20 mV. In vitro cell culture, and toxicity studies showed optimal use of these nanocarriers, with hemocompatibility data indicating non-toxicity. Ex vivo skin permeation analysis showed a skin permeation rate of 33% after 24 h. The optimized formulation was loaded in a carbopol-based gel that exhibits non-Newtonian flow characteristics with shear-thinning behavior and variable thixotropy. The nanoplexes delivered via gel demonstrated skin permeation of 57% after 24 h in mice skin ex vivo. In vivo skin toxicity testing confirmed the low toxicity profile of these nanocarriers. These results are promising for the transdermal/dermal delivery of macromolecules, such as protein therapeutics, using nanoplexes.

cRGD-modified hybrid lipopolymeric nanoplexes for gene editing in the posterior segment of the eye.

Eye-related diseases, specifically retinal dystrophy (RD) conditions, are the leading cause of blindness worldwide. Gene addition, regulation, or editing could potentially treat such diseases through gene expression regulation. CRISPR/Cas9 gene editing is one of the most prominent and precise gene editing tools which could be employed to edit genes related to the dystrophic condition. However, CRISPR/Cas9 faces in vivo delivery challenges due to its high molecular weight, negative charge, prone to degradation in the presence of nucleases and proteases, poor cellular degradation, etc., which makes it challenging to adopt for therapeutic applications. We developed cRGD-modified lipopolymeric nanoplexes loaded with Cas9 RNPs with a particle size and zeta potential of 175 ±â€¯20 nm and 2.15 ±â€¯0.9 mV, respectively. The cRGD-modified lipopolymeric nanoplexes were stable for 194 h and able to transfect >70 % ARPE-19 and NIH3T3 cells with an Indel frequency of ~40 % for the VEGF-A gene. The cRGD-modified lipopolymeric nanoplexes found good vitreous mobility and could transfection retinal cells in vivo after 48 h of intravitreal injection in Wistar Rats. Moreover, in vivo VEGFA gene editing was ~10 % with minimal toxicities. Collectively, the cRGD-modified lipopolymeric nanoplexes were found to have extreme potential in delivering CRISPR/Cas9 RNPs payload to the retinal tissues for therapeutic applications.

Temozolomide-fatty acid conjugates for glioblastoma multiforme: In vitro and in vivo evaluation.

Glioblastoma multiforme (GBM) is the deadliest brain tumor with a poor prognosis and limited therapeutic options. Temozolomide (TMZ) is the first-line chemotherapeutic agent used for the treatment of GBM; however, it suffers from several limitations, including short half-life, rapid metabolism, <1% brain bioavailability, methyl guanine methyl transferase (MGMT) based chemoresistance, and hematological toxicities. Several approaches have been adopted to overcome these limitations, particularly by using nanotechnology-based systems, but its physicochemical properties make TMZ challenging to load into these nanocarriers. In the current research, we conjugated TMZ with different fatty acids, i.e., linoleic acid (LA), oleic acid (OA), and palmitic acid (PA), to obtain TMZ-fatty acid conjugates, which are comparatively hydrophobic, less prone to degradation and potent. These conjugates were thoroughly characterized using 1H NMR spectroscopy, high-resolution mass spectrometry (HR-MS), and reverse phase-high performance liquid chromatography (RP-HPLC). The synthesized conjugates, namely Temozolomide-oleic acid (TOA,6R1), Temozolomide-linoleic acid (TLA, 6R2), and Temozolomide-palmitic acid (TPA, 6R3), showed an IC50 of 101.4, 67.97, and 672.04 µM, respectively in C6 cells and 428.257, 366.43 and 413.69 µM, respectively in U87-MG cells. On the other hand, the free TMZ showed an IC50 of >1000 µM and 564.23 µM in C6 and U87-MG, respectively. Further, the in vivo efficacy of the TMZ-fatty acid conjugates was evaluated in the C6-induced orthotropic rat glioblastoma model, wherein the TMZ-fatty acid conjugate showed improved survival rate (1.6 folds) and overall health of the animals. Collectively, the conjugation of fatty acids with TMZ improves its anticancer potential against glioblastoma multiforme (GBM).

Lipopolymeric Nanocarrier Enables Effective Delivery of CRISPR/Cas9 Expressing Plasmid.

CRISPR/Cas9 has proven its accuracy and precision for gene editing by making a double-strand break at the predetermined site. Despite being a mainstream gene editing tool, CRISPR/Cas9 has limitations for its in vivo delivery due to the physico-chemical properties such as high molecular weight, supranegative charge, degradation in the presence of nucleases, etc. Hereby, a cationic lipopolymer is explored for its efficiency in delivering CRISPR/Cas9 plasmid (pCas9) in vitro and in vivo. The lipopolymer is utilized to form blank cationic nanoplexes having a zeta potential of +15.8 ± 0.7 mV. Being cationic, the blank nanoplexes are able to condense the pCas9 plasmid at a ratio of 1:20 with a complexation efficiency of ≈98% and show a size and zeta potential of ≈141 ± 16 nm and 4.2 mV ± 0.7, respectively. The pCas9-loaded nanoplexes show a transfection efficiency of ≈69% in ARPE-19 cells and show ≈22% of indel frequency, indicating the successful translation of Cas9 protein and guide RNA in the cytosol. Further, they are found to be stable under in vivo environment when given intravenously in Swiss albino mice. These lipopolymeric nanoplexes can be a potential carrier for CRISPR plasmids for genome editing applications.

Polymeric and small molecule-conjugates of temozolomide as improved therapeutic agents for glioblastoma multiforme.

Temozolomide (TMZ), an imidazotetrazine, is a second-generation DNA alkylating agent used as a first-line treatment of glioblastoma multiforme (GBM). It was approved by FDA in 2005 and declared a blockbuster drug in 2008. Although TMZ has shown 100% oral bioavailability and crosses the blood-brain barrier effectively, however it suffers from limitations such as a short half-life (∼1.8 h), rapid metabolism, and lesser accumulation in the brain (∼10-20%). Additionally, development of chemoresistance has been associated with its use. Since it is a potential chemotherapeutic agent with an unmet medical need, advanced delivery strategies have been explored to overcome the associated limitations of TMZ. Nanocarriers including liposomes, solid lipid nanoparticles (SLNs), nanostructure lipid carriers (NLCs), and polymeric nanoparticles have demonstrated their ability to improve its circulation time, stability, tissue-specific accumulation, sustained release, and cellular uptake. Because of the appreciable water solubility of TMZ (∼5 mg/mL), the physical loading of TMZ in these nanocarriers is always challenging. Alternatively, the conjugation approach, wherein TMZ has been conjugated to polymers or small molecules, has been explored with improved outcomes in vitro and in vivo. This review emphasized the practical evidence of the conjugation strategy to improve the therapeutic potential of TMZ in the treatment of glioblastoma multiforme.

Oral sorafenib-loaded microemulsion for breast cancer: evidences from the in-vitro evaluations and pharmacokinetic studies.

Sorafenib tosylate (SFB) is a multikinase inhibitor that inhibits tumour growth and proliferation for the management of breast cancer but is also associated with issues like toxicity and drug resistance. Also, being a biopharmaceutical class II (BCS II) drug, its oral bioavailability is the other challenge. Henceforth, this report intended to encapsulate SFB into a biocompatible carrier with biodegradable components, i.e., phospholipid. The microemulsion of the SFB was prepared and characterized for the surface charge, morphology, micromeritics and drug release studies. The cell viability assay was performed on 4T1 cell lines and inferred that the IC50 value of sorafenib-loaded microemulsion (SFB-loaded ME) was enhanced compared to the naïve SFB at the concentrations of about 0.75 µM. More drug was available for the pharmacological response, as the protein binding was notably decreased, and the drug from the developed carriers was released in a controlled manner. Furthermore, the pharmacokinetic studies established that the developed nanocarrier was suitable for the oral administration of a drug by substantially enhancing the bioavailability of the drug to that of the free SFB. The results bring forth the preliminary evidence for the future scope of SFB as a successful therapeutic entity in its nano-form for effective and safer cancer chemotherapy via the oral route.

Opportunities and challenges of fatty acid conjugated therapeutics.

Instability, poor cellular uptake and unfavorable pharmacokinetics and biodistribution of many therapeutic molecules require modification in their physicochemical properties. The conjugation of these APIs with fatty acids has demonstrated an enhancement in their lipophilicity and stability. The improvement in the formulations that resulted from the conjugation of a drug with a fatty acid includes increased half-life, enhanced cellular uptake and retention, targeted tumor delivery, reduced chemoresistance in cancer, and improved blood-brain-barrier (BBB) penetration. In this review, various therapeutic molecules, including small molecules, peptides and oligonucleotides, that have been conjugated with fatty acid have been thoroughly discussed along with various conjugation strategies. The application of nano-system based delivery is gaining a lot of attention due to its ability to provide controlled drug release, targeting and reducing the extent of side effects. This review also covers various nano-carriers that have been utilized for the delivery of fatty acid drug conjugates. The enhanced lipophilicity of the drug-fatty acid conjugate has shown to enhance the affinity of the drug towards these carriers, thereby increasing the entrapment efficiency and formulation performance.