When it is too much: Identifying butamben excess on the surface of pharmaceutical preformulation samples by Raman mapping.

Butamben is a topical local anesthetic which formulation in lipid-based drug delivery systems (DDS) is challenging due to its affinity for hydrophilic excipients. This means that a medium polarity excipient is preferred for the development of a stable nanostructured lipid carrier (NLC) formulation. In turn, in NLC, the type and number of excipients will determine the active pharmaceutical ingredient (API) solubility and the maximum drug upload. To solve this dilemma and get the best formulation, a throughout screening study to evaluate API solubilization in different excipients was carried out. Subsequently, excipients with different solubilization capacities were selected for microscopic evaluation by Raman mapping, and in turn analysis of the distributional homogeneity index (DHI) and standard deviation of the histograms allowed solving the posed question. Design of experiments (DoE) was employed to understand better the interactions between the excipients; linear and higher-order models were obtained with R2 above of 0.8824. Even though DHI is a good parameter to be used as response, an API concentration higher than 30% (w/w) provided a homogeneous surface in case of good miscibility and, in this case, this parameter needs to be employed with an inspection and/or evaluation of other parameters. A curve of concentration vs. mean scores of images proved to be an alternative to identify the saturation/limit of linear range.

Neither too little nor too much: Finding the ideal proportion of excipients using confocal Raman and chemometrics.

The applications of Raman imaging in pharmaceutical field are ever-increasing due its ability to obtain spatial and spectral information simultaneously, once it allows determine the chemical distribution of compounds. In this sense, it is used to study homogeneity, of paramount importance during the development of pharmaceutical formulations due to its relation to stability, safety and efficacy. Commonly, just surface is analyzed, but confocal Raman spectroscopy can also characterize the inner part of samples, allowing to determine phase separation in the early stages. In this sense, confocal 3D Raman microscopy was crucial to obtain the optimal proportion of Apifil®, Capryol® 90 and Transcutol® to promote controlled release of the local anesthetic butamben (BTB). 3D chemical maps were obtained by classical least squares (CLS) using pure compound spectra as S matrix, showing that chemical distribution throughout the material was different. Knowing that the composition of samples affects the homogeneity parameter, standard deviation and distributional homogeneity index (DHI) were used in mixture experimental design (DoE). From this analysis, it was revealed that a correct amount of Capryol® 90 enhances both miscibility and solubility. Furthermore, suitable miscibility was observed in two ratio proportions of excipients with a desirability of 0.783 and 0.742. These results unequivocally demonstrated that confocal Raman microscopy combined to DoE can bring pharmaceutical development to a higher level.

A pre-formulation study of tetracaine loaded in optimized nanostructured lipid carriers.

Tetracaine (TTC) is a local anesthetic broadly used for topical and spinal blockade, despite its systemic toxicity. Encapsulation in nanostructured lipid carriers (NLC) may prolong TTC delivery at the site of injection, reducing such toxicity. This work reports the development of NLC loading 4% TTC. Structural properties and encapsulation efficiency (%EE > 63%) guided the selection of three pre-formulations of different lipid composition, through a 23 factorial design of experiments (DOE). DLS and TEM analyses revealed average sizes (193-220 nm), polydispersity (< 0.2), zeta potential |- 21.8 to - 30.1 mV| and spherical shape of the nanoparticles, while FTIR-ATR, NTA, DSC, XRD and SANS provided details on their structure and physicochemical stability over time. Interestingly, one optimized pre-formulation (CP-TRANS/TTC) showed phase-separation after 4 months, as predicted by Raman imaging that detected lack of miscibility between its solid (cetyl palmitate) and liquid (Transcutol) lipids. SANS analyses identified lamellar arrangements inside such nanoparticles, the thickness of the lamellae been decreased by TTC. As a result of this combined approach (DOE and biophysical techniques) two optimized pre-formulations were rationally selected, both with great potential as drug delivery systems, extending the release of the anesthetic (> 48 h) and reducing TTC cytotoxicity against Balb/c 3T3 cells.

Docetaxel and Lidocaine Co-Loaded (NLC-in-Hydrogel) Hybrid System Designed for the Treatment of Melanoma.

Melanoma is the most aggressive skin carcinoma and nanotechnology can bring new options for its pharmacological treatment. Nanostructured lipid carriers (NLC) are ideal drug-delivery carriers for hydrophobic drugs, such as the antineoplastic docetaxel (DTX), and hybrid (NLC-in-hydrogel) systems are suitable for topical application. This work describes a formulation of NLCDTX in xanthan-chitosan hydrogel containing lidocaine (LDC) with anticancer and analgesia effects. The optimized nanoparticles encapsulated 96% DTX and rheological analysis revealed inherent viscoelastic properties of the hydrogel. In vitro assays over murine fibroblasts (NIH/3T3) and melanoma cells (B16-F10), human keratinocytes (HaCaT) and melanoma cells (SK-MEL-103) showed reduction of docetaxel cytotoxicity after encapsulation in NLCDTX and HGel-NLCDTX. Addition of LDC to the hybrid system (HGel-NLCDTX-LDC) increased cell death in tumor and normal cells. In vivo tests on C57BL/6J mice with B16-F10-induced melanoma indicated that LDC, NLCDTX, HGel-NLCDTX-LDC and NLCDTX + HGel-LDC significantly inhibited tumor growth while microPET/SPECT/CT data suggest better prognosis with the hybrid treatment. No adverse effects were observed in cell survival, weight/feed-consumption or serum biochemical markers (ALT, AST, creatinine, urea) of animals treated with NLCDTX or the hybrid system. These results confirm the adjuvant antitumor effect of lidocaine and endorse HGel-NLCDTX-LDC as a promising formulation for the topical treatment of melanoma.

Cellulose Nanocrystals/Chitosan-Based Nanosystems: Synthesis, Characterization, and Cellular Uptake on Breast Cancer Cells.

Cellulose nanocrystals (CNCs) are elongated biobased nanostructures with unique characteristics that can be explored as nanosystems in cancer treatment. Herein, the synthesis, characterization, and cellular uptake on folate receptor (FR)-positive breast cancer cells of nanosystems based on CNCs and a chitosan (CS) derivative are investigated. The physical adsorption of the CS derivative, containing a targeting ligand (folic acid, FA) and an imaging agent (fluorescein isothiocyanate, FITC), on the surface of the CNCs was studied as an eco-friendly methodology to functionalize CNCs. The fluorescent CNCs/FA-CS-FITC nanosystems with a rod-like morphology showed good stability in simulated physiological and non-physiological conditions and non-cytotoxicity towards MDA-MB-231 breast cancer cells. These functionalized CNCs presented a concentration-dependent cellular internalization with a 5-fold increase in the fluorescence intensity for the nanosystem with the higher FA content. Furthermore, the exometabolic profile of the MDA-MB-231 cells exposed to the CNCs/FA-CS-FITC nanosystems disclosed a moderate impact on the cells' metabolic activity, limited to decreased choline uptake and increased acetate release, which implies an anti-proliferative effect. The overall results demonstrate that the CNCs/FA-CS-FITC nanosystems, prepared by an eco-friendly approach, have a high affinity towards FR-positive cancer cells and thus might be applied as nanocarriers with imaging properties for active targeted therapy.

Lipid nanoparticles loaded with butamben and designed to improve anesthesia at inflamed tissues.

The most frequently used local anesthetics (LA) for local infiltration have an ionizable amine in the range of pH 7.6-8.9. Effective anesthesia of inflamed tissues is a great challenge, especially because the induced local acidosis decreases the fraction of the neutral (more potent) LA species in situ. To solve this limitation, the butyl-substituted benzocaine analogue butamben (BTB) - that has no ionizable amine group close to the physiological pH - could be useful if it was not for its low solubility. To overcome the solubility problem, an optimized formulation for BTB using nanostructured lipid carriers (NLC) was developed by a factorial design and characterized using DLS, XRD, DSC and cryo-EM. The release kinetics and cytotoxicity of the new formulation were measured in vitro, while the in vivo tests assessed its effectiveness on healthy and inflamed tissues, in rats. The optimized NLCBTB formulation showed desirable physicochemical properties (size = 235.6 ± 3.9 nm, polydispersity = 0.182 ± 0.006 and zeta potential = -23.6 ± 0.5 mV), high (99.5%) encapsulation efficiency and stability during 360 days of storage at room temperature. NLCBTB prolonged the release of butamben and decreased its in vitro cytotoxicity without inducing any in vivo toxic alteration. In the inflammatory hyperalgesia model, the NLCBTB formulation showed potential for the management of inflammatory pain, displaying greater analgesic effectiveness (40%) and a prolonged effect.

Functional Hybrid Nanoemulsions for Sumatriptan Intranasal Delivery.

In recent years, advanced nanohybrid materials processed as pharmaceuticals have proved to be very advantageous. Triptans, such as the commercially available intranasal sumatriptan (SMT), are drugs employed in the treatment of painful migraine symptoms. However, SMT effectiveness by the intranasal route is limited by its high hydrophilicity and poor mucoadhesion. Therefore, we designed hybrid nanoemulsions (NE) composed of copaiba oil as the organic component plus biopolymers (xanthan, pectin, alginate) solubilized in the continuous aqueous phase, aiming at the intranasal release of SMT (2% w/v). Firstly, drug-biopolymer complexes were optimized in order to decrease the hydrophilicity of SMT. The resultant complexes were further encapsulated in copaiba oil-based nanoparticles, forming NE formulations. Characterization by FTIR-ATR, DSC, and TEM techniques exposed details of the molecular arrangement of the hybrid systems. Long-term stability of the hybrid NE at 25°C was confirmed over a year, regarding size (~ 120 nm), polydispersity (~ 0.2), zeta potential (~ -25 mV), and nanoparticle concentration (~ 2.1014 particles/mL). SMT encapsulation efficiency in the formulations ranged between 41-69%, extending the in vitro release time of SMT from 5 h (free drug) to more than 24 h. The alginate-based NE was selected as the most desirable system and its in vivo nanotoxicity was evaluated in a zebrafish model. Hybrid NE treatment did not affect spontaneous movement or induce morphological changes in zebrafish larvae, and there was no evidence of mortality or cardiotoxicity after 48 h of treatment. With these results, we propose alginate-based nanoemulsions as a potential treatment for migraine pain.

Articaine in functional NLC show improved anesthesia and anti-inflammatory activity in zebrafish.

Anesthetic failure is common in dental inflammation processes, even when modern agents, such as articaine, are used. Nanostructured lipid carriers (NLC) are systems with the potential to improve anesthetic efficacy, in which active excipients can provide desirable properties, such as anti-inflammatory. Coupling factorial design (FD) for in vitro formulation development with in vivo zebrafish tests, six different NLC formulations, composed of synthetic (cetyl palmitate/triglycerides) or natural (avocado butter/olive oil/copaiba oil) lipids were evaluated for loading articaine. The formulations selected by FD were physicochemically characterized, tested for shelf stability and in vitro release kinetics and had their in vivo effect (anti-inflammatory and anesthetic effect) screened in zebrafish. The optimized NLC formulation composed of avocado butter, copaiba oil, Tween 80 and 2% articaine showed adequate physicochemical properties (size = 217.7 ± 0.8 nm, PDI = 0.174 ± 0.004, zeta potential = - 40.2 ± 1.1 mV, %EE = 70.6 ± 1.8) and exhibited anti-inflammatory activity. The anesthetic effect on touch reaction and heart rate of zebrafish was improved to 100 and 60%, respectively, in comparison to free articaine. The combined FD/zebrafish approach was very effective to reveal the best articaine-in-NLC formulation, aiming the control of pain at inflamed tissues.

Hybrid nanofilms as topical anesthetics for pain-free procedures in dentistry.

Topical anesthetics are widely applied in order to relieve the discomfort and anxiety caused by needle insertion and other painful superficial interventions at the oral cavity. So far, there are no commercially available effective topical anesthetic formulations for that purpose, and the most of developments are related to hydrophilic and low mucoadhesive forms. Therefore, we have prepared different hybrid nanofilms composed of biopolymer matrices (chitosan, pectin, and chitosan-pectin) blended with nanostructured lipid carriers (NLC) loading the eutectic mixture of 5% lidocaine-prilocaine (LDC-PLC), in order to fulfill this gap in the market. These dual systems were processed as hybrid nanofilms by the solvent/casting method, and its mucoadhesive, structural and mechanical properties were detailed. The most appropriate hybrid nanofilm combined the advantages of both pectin (PCT) and NLC components. The resultant material presented sustained LDC-PLC release profile for more than 8 h; permeation across porcine buccal mucosa almost twice higher than control and non-cytotoxicity against 3T3 and HACAT cell lines. Then, the in vivo efficacy of PCT/NLC formulation was compared to biopolymer film and commercial drug, exhibiting the longest-lasting anesthetic effect (> 7 h), assessed by tail flick test in mice. These pectin-based hybrid nanofilms open perspectives for clinical trials and applications beyond Dentistry.

Injectable in situ forming nanogel: A hybrid Alginate-NLC formulation extends bupivacaine anesthetic effect.

Finding an ideal anesthetic agent for postoperative pain control, with long action and low side effects, is still a challenge. Local anesthetics have potential for such application if their time of action is improved. This work introduces a new hybrid formulation formed by the association of a nanostructured lipid carrier with a biopolymeric system to encapsulate bupivacaine (BVC). The hybrid formulation was physicochemical and structurally characterized by DLS, TEM, DSC, XRD and FTIR-ATR, and it remained stable for 12 months at room temperature. In vivo analgesia and imaging tests showed that the hybrid system was able to modulate the release, and to increase the concentration of BVC at the site of action, by forming a nanogel in situ. Such nanogel improved over 5 times (>24 h) the anesthesia duration, when compared to free BVC at clinical (0.5%) doses. Therefore, this novel in situ-forming nanogel shows great potential to be used in postsurgical pain control, improving the action of BVC, without losing its versatility of (infiltrative) application.

Improved efficacy of naproxen-loaded NLC for temporomandibular joint administration.

Inflammatory conditions of the temporomandibular joint (TMJ) and peripheral tissues affect many people around the world and are commonly treated with non-steroidal anti-inflammatory drugs (NSAIDs). However, in order to get desirable results, treatments with NSAIDs may take weeks, causing undesirable side effects and requiring repeated administration. In this sense, this work describes the development of an optimized nanostructured lipid carrier (NLC) formulation for intra-articular administration of naproxen (NPX). An experimental design (23) selected the best formulation in terms of its physicochemical and structural properties, elucidated by different methods (DLS, NTA, TEM, DSC, and ATR-FTIR). The chosen formulation (NLC-NPX) was tested on acute inflammatory TMJ nociception, in a rat model. The optimized excipients composition provided higher NPX encapsulation efficiency (99.8%) and the nanoparticles were found stable during 1 year of storage at 25 °C. In vivo results demonstrated that the sustained delivery of NPX directly in the TMJ significantly reduced leukocytes migration and levels of pro-inflammatory cytokines (IL-1ß and TNF-α), for more than a week. These results point out the NLC-NPX formulation as a promising candidate for the safe treatment of inflammatory pain conditions of TMJ or other joints.

Evaluation of miscibility and polymorphism of synthetic and natural lipids for nanostructured lipid carrier (NLC) formulations by Raman mapping and multivariate curve resolution (MCR).

Nanostructured lipid carriers (NLC) belong to youngest lipid-based nanocarrier class and they have gained increasing attention over the last ten years. NLCs are composed of a mixture of solid and liquid lipids, which solubilizes the active pharmaceutical ingredient, stabilized by a surfactant. The miscibility of the lipid excipients and structural changes (polymorphism) play an important role in the stability of the formulation and are not easily predicted in the early pharmaceutical development. Even when the excipients are macroscopically miscible, microscopic heterogeneities can result in phase separation during storage, which is only detected after several months of stability studies. In this sense, this work aimed to evaluate the miscibility and the presence of polymorphism in lipid mixtures containing synthetic (cetyl palmitate, Capryol 90®, Dhaykol 6040 LW®, Precirol ATO5® and myristyl myristate) and natural (beeswax, cocoa and shea butters, copaiba, sweet almond, sesame and coconut oils) excipients using Raman mapping and multivariate curve resolution - alternating least squares (MCR-ALS) method. The results were correlated to the macroscopic stability of the formulations. Chemical maps constructed for each excipient allowed the direct comparison among formulations, using standard deviation of the histograms and the Distributional Homogeneity Index (DHI). Lipid mixtures of cetyl palmitate/Capryol®; cetyl palmitate/Dhaykol®; myristyl myristate/Dhaykol® and myristyl myristate/coconut oil presented a single histogram distribution and were stable. The sample with Precirol®/Capryol® was not stable, although the histogram distribution was narrower than the samples with cetyl palmitate, indicating that miscibility was not the factor responsible for the instability. Structural changes before and after melting were identified for cocoa butter and shea butter, but not in the beeswax. Beeswax + copaiba oil sample was very homogenous, without polymorphism and stable over 6 months. Shea butter was also homogeneous and, in spite of the polymorphism, was stable. Formulations with cocoa butter presented a wider histogram distribution and were unstable. This paper showed that, besides the miscibility evaluation, Raman imaging could also identify the polymorphism of the lipids, two major issues in lipid-based formulation development that could help guide the developer understand the stability of the NLC formulations.

Optimised NLC: a nanotechnological approach to improve the anaesthetic effect of bupivacaine.

The short time of action and systemic toxicity of local anaesthetics limit their clinical application. Bupivacaine is the most frequently used local anaesthetic in surgical procedures worldwide. The discovery that its S(-) enantiomeric form is less toxic than the R(+) form led to the introduction of products with enantiomeric excess (S75:R25 bupivacaine) in the market. Nevertheless, the time of action of bupivacaine is still short; to overcome that, bupivacaine S75:R25 (BVCS75) was encapsulated in nanostructured lipid carriers (NLC). In this work, we present the development of the formulation using chemometric tools of experimental design to study the formulation factors and Raman mapping associated with Classical Least Squares (CLS) to study the miscibility of the solid and the liquid lipids. The selected formulation of the nanostructured lipid carrier containing bupivacaine S75:R25 (NLCBVC) was observed to be stable for 12 months under room conditions regarding particle size, polydispersion, Zeta potential and encapsulation efficiency. The characterisation by DSC, XDR and TEM confirmed the encapsulation of BVCS75 in the lipid matrix, with no changes in the structure of the nanoparticles. The in vivo analgesic effect elicited by NLCBVC was twice that of free BVCS75. Besides improving the time of action, no statistical difference in the blockage of the sciatic nerve of rats was found between 0.125% NLCBVC and 0.5% free BVCS75. Therefore, the formulation allows a reduction in the required anaesthesia dose, decreasing the systemic toxicity of bupivacaine, and opening up new possibilities for different clinical applications.