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.

Mucoadhesive, Thermoreversible Hydrogel, Containing Tetracaine-Loaded Nanostructured Lipid Carriers for Topical, Intranasal Needle-Free Anesthesia.

Recent advances have been reported for needle-free local anesthesia in maxillary teeth by administering a nasal spray of tetracaine (TTC) and oxymetazoline, without causing pain, fear, and stress. This work aimed to assess whether a TTC-loaded hybrid system could reduce cytotoxicity, promote sustained permeation, and increase the anesthetic efficacy of TTC for safe, effective, painless, and prolonged analgesia of the maxillary teeth in dental procedures. The hybrid system based on TTC (4%) encapsulated in nanostructured lipid carriers (NLC) and incorporated into a thermoreversible hydrogel of poloxamer 407 (TTCNLC-HG4%) displayed desirable rheological, mechanical, and mucoadhesive properties for topical application in the nasal cavity. Compared to control formulations, the use of TTCNLC-HG4% slowed in vitro permeation of the anesthetic across the nasal mucosa, maintained cytotoxicity against neuroblastoma cells, and provided a three-fold increase in analgesia duration, as observed using the tail-flick test in mice. The results obtained here open up perspectives for future clinical evaluation of the thermoreversible hybrid hydrogel, which contains TTC-loaded NLC, with the aim of creating an effective, topical, intranasal, needle-free anesthesia for use in dentistry.

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.

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.

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.

Comparison of different chemometric methods to extract chemical and physical information from Raman images of homogeneous and heterogeneous semi-solid pharmaceutical formulations.

In formulations of nanostructured lipid carriers, lipid solid dispersions and self-emulsifying drug delivery systems, it is common that a solid or semi-solid lipid excipient is mixed with a liquid solvent or liquid lipid. Even when the excipients are visually miscible upon melting, they might have microscopic non-homogeneities which could lead to instability over time and future phase separation. Raman mapping associated with chemometric methods can be useful to evaluate spatial distribution of compounds, however it has not been extensively applied to the formulations mentioned above. The aim of this work was to compare the outcomes of three different chemometric methods - principal components analysis (PCA), multivariate curve resolution with alternating least squares (MCR-ALS) and independent components analysis (ICA) - to study two systems of very different degrees of microscopic miscibility: cetyl palmitate + Transcutol© (heterogeneous) and polyethylene glycol 6000 (PEG 6000) + Tween 80© (homogeneous). These two samples were chosen due to large differences in spatial distribution of the compounds over the pixels which could require different approaches for data treatment. The three methods were compared regarding recovered concentrations (or scores), signals (or loadings) and the need for matrix augmentation to obtain reliable results. Results showed that PCA loadings were the mathematical differences of the spectra of pure compounds for both samples, and therefore only 'contrast images' could be generated. MCR and ICA provided signals that could be related to the chemical components, however MCR presented rotational ambiguities even for the very heterogeneous sample, a situation in which ICA performed better as a blind search method. For the homogeneous sample, both methods showed rank deficiency and therefore the use of a matrix augmentation was necessary. ICA and PCA allowed identifying physical modifications in the homogeneous semi-solid PEG 6000/Tween 80® sample over the time, probably due to the folding/unfolding of the crystalline chains of PEG 6000. Therefore, this work discusses the ability of the three chemometrics methods to extract information from Raman spectra in order to characterize the chemical, spatial and even physical aspects of semi-solid pharmaceutical formulations, which could be of much use for stability studies of different drug delivery systems.

Dibucaine in Ionic-Gradient Liposomes: Biophysical, Toxicological, and Activity Characterization.

Administration of local anesthetics is one of the most effective pain control techniques for postoperative analgesia. However, anesthetic agents easily diffuse into the injection site, limiting the time of anesthesia. One approach to prolong analgesia is to entrap local anesthetic agents in nanostructured carriers (e.g., liposomes). Here, we report that using an ammonium sulphate gradient was the best strategy to improve the encapsulation (62.6%) of dibucaine (DBC) into liposomes. Light scattering and nanotracking analyses were used to characterize vesicle properties, such as, size, polydispersity, zeta potentials, and number. In vitro kinetic experiments revealed the sustained release of DBC (50% in 7 h) from the liposomes. In addition, in vitro (3T3 cells in culture) and in vivo (zebrafish) toxicity assays revealed that ionic-gradient liposomes were able to reduce DBC cyto/cardiotoxicity and morphological changes in zebrafish larvae. Moreover, the anesthesia time attained after infiltrative administration in mice was longer with encapsulated DBC (27 h) than that with free DBC (11 h), at 320 µM (0.012%), confirming it as a promising long-acting liposome formulation for parenteral drug administration of DBC.

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.

Natural lipids-based NLC containing lidocaine: from pre-formulation to in vivo studies.

In a nanotechnological approach we have investigated the use of natural lipids in the preparation of nanostructured lipid carriers (NLC). Three different NLC composed of copaiba oil and beeswax, sweet almond oil and shea butter, and sesame oil and cocoa butter as structural matrices were optimized using factorial analysis; Pluronic® 68 and lidocaine (LDC) were used as the colloidal stabilizer and model encapsulated drug, respectively. The optimal formulations were characterized by different techniques (IR-ATR, DSC, and TEM), and their safety and efficacy were also tested. These nanocarriers were able to upload high amounts of the anesthetic with a sustained in vitro release profile for 24h. The physicochemical stability in terms of size (nm), PDI, zeta potential (mV), pH, nanoparticle concentration (particles/mL), and visual inspection was followed during 12months of storage at 25°C. The formulations exhibited excellent structural properties and stability. They proved to be nontoxic in vitro (cell viability tests with Balb/c 3T3 fibroblasts) and significantly improved the in vivo effects of LDC, over the heart rate of zebra fish larvae and in the blockage of sciatic nerve in mice. The results from this study support that the proper combination of natural excipients is promising in DDS, taking advantage of the biocompatibility, low cost, and diversity of lipids.

Nanostructured lipid carriers as robust systems for topical lidocaine-prilocaine release in dentistry.

In dental practice, local anesthesia causes pain, fear, and stress, and is frequently the reason that patients abandon treatment. Topical anesthetics are applied in order to minimize the discomfort caused by needle insertion and injection, and to reduce the symptoms of superficial trauma at the oral mucosa, but there are still no efficient commercially available formulations. Factorial design is a multivariate data analysis procedure that can be used to optimize the manufacturing processes of lipid nanocarriers, providing valuable information and minimizing development time. This work describes the use of factorial design to optimize a process for the preparation of nanostructured lipid carriers (NLC) based on cetyl palmitate and capric/caprylic triglycerides as structural lipids and Pluronic 68 as the colloidal stabilizer, for delivery of the local anesthetics lidocaine and prilocaine (both at 2.5%). The factors selected were the excipient concentrations, and three different responses were followed: particle size, polydispersity index and zeta potential. The encapsulation efficiency of the most effective formulations (NLC 2, 4, and 6) was evaluated by the ultrafiltration/centrifugation method. The formulations that showed the highest levels of encapsulation were tested using in vitro release kinetics experiments with Franz diffusion cells. The NLC6 formulation exhibited the best sustained release profile, with 59% LDC and 66% PLC released after 20h. This formulation was then characterized using different techniques (IR-ATR, DSC, DRX, TEM, and NTA) to obtain information about its molecular organization and its physicochemical stability, followed during 14months of storage at 25°C. This thorough pre-formulation study represents an important advance towards the development of an efficient pre-anesthetic for use in dentistry.