Nanoencapsulation of general anaesthetics

General anaesthetics are routinely used to sedate patients during prolonged surgeries and administered via intravenous injection and/or inhalation. All anaesthetics have short half-lives, hence the need for their continuous administration. This causes several side effects such as pain, vomiting, nausea, bradycardia, and on rare occasions death post-administration. Several clinical trials studied the synergetic effect of a combination of anaesthetic drugs to reduce the drug load. Another solution is to encapsulate anaesthetics in nanoparticles to reduce their dose and side effects as well as achieve their sustained release manner. Different types of nanoparticles were developed as carriers of intravenous and intrathecal anaesthetics generating platforms which facilitate drug transport across the blood–brain barrier (BBB). Nanocarriers encapsulating common anaesthetic drugs such as propofol, etomidate, and ketamine were developed and characterized in terms of size, stability, onset and duration of loss of right reflex, and tolerance to pain in small animal models. The review discusses the types of nanocarriers used to reduce the side effects of the anaesthetic drugs while prolonging the sedation time. More rigorous studies are still required to evaluate the nanocarrier formulations regarding their ability to deliver anaesthetic drugs across the BBB, safety, and finally applicability in clinical settings.


Introduction
General anaesthesia, controlled loss of consciousness (LOC), is necessary to perform prolonged surgeries. 1General anaesthetic drugs are introduced intravenously to patients, at the beginning of surgery, to induce a rapid onset of sedation.Due to the short half-life of the drugs, sedation is maintained via further intravenous administration of anaesthetics or introduction of volatile anaesthetics (inhaled by the patient). 2Prolonged administration of anaesthetic drugs, however, causes several side effects, which may harm patients post-surgery, including hypotension, bradycardia, surgical infection, and Basma M: T: Abdoullateef Basma Mohamed Tarek Abdoullateef received her BSc in medicine from faculty of medicine, Zagazig University.She worked as a specialist in microbiology and immunology in Theodor Bilharz Research Institute, Cairo, Egypt, where she developed her microbiological laboratory skills and medical knowledge.She received her Msc degree in medical microbiology and immunology from the faculty of medicine, Cairo University in May 2023.Currently, Basma is working as a research assistant and is a MSc student in the biotechnology program, School of Science and Engineering, the American University in Cairo, Egypt under the supervision of Dr Rania Siam.

Saif El-Din Al-Mofty
Saif El-Din El-Moy received his BSc in Biomedical Science from the University of Science & Technology, Zewail City.He was a lab specialist at the Centre of Material Science, where he developed nanobers for healing burns and microgels loaded with doxorubicin for breast cancer.Currently, Saif is working as a research assistant studying the antimicrobial, and cytotoxic effects of metal-oxide nanoparticles, hemostatic sponges, and natural extracts at the department of chemistry, the American University in Cairo under the supervision of Dr Hassan Azzazy.thromboembolism. 3For selected surgeries, intrathecal or epidural anaesthesia were introduced to avoid unnecessary general anaesthesia. 4However, these routes aren't suitable for majority of surgeries such as open heart, tumour excision, and brain surgeries.
The blood-brain barrier (BBB) is made up of endothelial cells (bound together via tight junctions), pericytes, and astrocytes (Fig. 1).It regulates entry of metabolites and nutrients but prevents entry of harmful substances into the brain in order to maintain a suitable environment for proper function of neurons. 5Small (<500 Da) and hydrophobic molecules can pass the BBB via simple diffusion while hydrophilic or large molecules in the circulation can pass through active transport and transcytosis. 6Many strategies have been developed to deliver drugs and anaesthetics across the BBB.
Physiochemical characteristics of drugs can be manipulated by loading into different nanocarrier systems to target drugs to specic tissues, improve drug bioavailability and biocompatibility, and achieve controlled release. 8,9Inorganic and organic nanocarriers were developed for drug delivery which demonstrated great potential to pass the BBB.0][11][12] Polymeric nanoparticles were employed for delivery of dopamine to treat Parkinson disease, 8 asialo-erythropoietin-modied PEGylated liposomes were used for treating cerebral ischemia-reperfusion injury, 13 and PLGA/ polysorbate-80 nanoparticles were delivered to brain tissues via the carotid artery. 14Dendrimers were used for delivery of drugs for treatment of cerebral palsy and other neuroinammatory disorders 15 and doxorubicin was encapsulated in stearic acid-  graed chitosan micelles as a promising brain-targeting delivery system. 16Ultrasmall gold nanoparticles (2 nm) surface-modied with uoresceine were shown to cross the BBB. 17Additionally, magnetic nanoparticles coated with heparin were shown to effectively protect neurons from the harmful effect of the accumulation of b amyloid, a hallmark of Alzheimer's disease. 18Finally, multiwalled carbon nanotubes were modied with angiopep-2 and used for the transport of anticancer drugs to treat brain glioma. 195][26] Additionally, most anaesthetic drugs are lipophilic which makes delivering these drugs via intravenous route difficult.4][25][26][27][28][29][30][31][32][33][34][35][36][37] LOC time is largely dependent on the dose administered and bioavailability of the drug which are also responsible for the known side effects.
Therefore, nanoformulations to encapsulate anaesthetic drugs were designed to increase bioavailability, and biodistribution in the brain, lower their dose while prolonging the sedation time and decreasing their side effects.Several studies reported the use of different nanocarriers to encapsulate propofol, ketamine, and etomidate (Fig. 2).Tests used to assess the effects of different anaesthetics in animals include loss of righting reex (LORR) and paw-licking test.LORR is a method to test the onset of loss of consciousness from the time of administration of the anaesthetic drug. 28,30,33,37The paw-licking test is used to study pain inicted on the rat during and aer administration.In this review, the platforms used for nanoencapsulation of general anaesthetics will be summarized with a focus on their particle size, encapsulation efficiency, ability to induce LORR and analgesia.

Propofol
Propofol (Diprivan, Lipuro) is a lipophilic anaesthetic agent which is administered continuously during prolonged surgeries.It is usually composed of 10 mg mL −1 of propofol emulsied in soybean oil, glycerol, and egg lecithin.Propofol has been reported to maintain a LORR from 3 to 13 min and a half-life of 0.9 h. 28,30,33,37Although propofol induces rapid loss of consciousness, patients recover from the sedation state quickly.9][40] Therefore, several studies have explored alternative vehicles to overcome such obstacles for propofol encapsulation (Table 1).

Encapsulation of propofol in lipid nanoparticles
Lipid nanoparticles can bypass the BBB and release their anaesthetic cargo in a slow manner which could prolong the LORR effect.They are prepared of a lipid moiety and a stabilizer of varying molecular sizes such as polysorbate 80 or Tween 80.2][43] Johnson et al., used soy phosphatidylcholine, glycerol dioleate, and polysorbate to create a lipid crystal nano emulsion with a particle size around 118 nm. 33The propofol

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concentration in this nano formula was 10 mg mL −1 (which is similar to propofol concentration in commercial Lipuro) which exhibited a LORR duration of 7.5 min compared to 6.3 min for Lipuro.However, propofol in lipid nanocrystal had a longer half-life time of 0.9 h as compared to 2.9 h for Lipuro in Sprague Dawley rats. 34This was due to the clearance of the propofol in the lipid nanocrystal was almost half (2799 mL h −1 ) that of Lipuro (4326 mL h −1 ).The study, however, didn't explore the pain  30 A third lipid-free nanoformulation was also prepared and named propofol nano sponge (PNS).ProNano demonstrated a lower free propofol concentration (0.13%) which reduced injection pain as compared to Diprivan.ProNano and PSNE showed LORR duration of 14 min and 15 min, respectively, as compared to LORR of 13 min for Diprivan.Additionally, the lipid content of the medium-chain glyceride-based nano emulsion (ProNano) was low, potentially lowering the risk of hyperlipidemia (a Diprivan side effect).On the other hand, PNS demonstrated a longer LORR duration of 21 min which may be related to the sustained release feature of PNS. 30 On the other hand, paw licking was decreased in animals administered propofol in lipid nanoparticles as compared to those which received Diprivan which indicates low pain induction upon injection.Propofol nanoencapsulation may reduce susceptibility to infection related to propofol immunomodulatory effects. 30ore studies, however, are needed to evaluate the systemic biodistribution of nano encapsulated propofol especially in the CNS and assess its potential in reducing susceptibility to microbial infection.

Palmitoyl quaternary ammonium glycol chitosan nanoparticles
Quaternary ammonium chitosan (QCS) is prepared by replacing amine group of chitosan with quaternary structures forming a potent positive charge within the chitosan molecule.QCS is water soluble regardless of the pH.However, to load propofol, a hydrophobic moiety would be needed.For this purpose, two studies employed palmitoyl oil or dendrimer core and showed similar entrapment efficiency and small nanoparticle size. 28,44However, the dendrimer core resulted in variation in nanoparticle size of QCS, PDI >0.6 (Table 1).Despite their poor homogeneity, the dendrimer core QCS exhibited a lower LORR in mice upon administration when compared to Diprivan. 28,44ginate graed nanoparticles Alginate nanoparticles are used for drug delivery for several reasons: biodegradability, enhanced bioavailability of the drug, and ability to encapsulate hydrophobic drugs with great stability.Propofol was encapsulated in alginate nanoparticles graed with octanol.32 The encapsulation efficiency was 99% with a nanoparticle size of 80 nm as evident by TEM.The nanoparticles developed also achieved sustained release of 20% in 2 h and 72% in 48 h with an onset of LORR of 35 s.This study demonstrated a promising nanoparticle delivery system but lacked the safety studies of haemolysis for octanol-alginate, pain response, and its biodistribution.It is of note, however, that sodium alginate was reported to have a generally low haemolytic activity.45

Propionylated amylose nanoparticles
Propionic acid is the smallest fatty acid which can attach to phosphatidylethanolamine found in the endothelial cells of the BBB. 25 A hydrophobic system was developed using propionylated amylose (PPA) to post-load propofol and actively target propofol to the BBB (Fig. 3).The particle size of the PPA was 55 nm.The propofol in PPA reached the brain in 3 min and was persistent for 30 min, then the PPA started to distribute through the liver, lungs and nally to the kidneys.The PPA developed reduced the dose needed for propofol to induce LORR and even induced a faster LORR onset time of up to 51 s.However, the LORR was kept for only 5 min as the nanoparticle exhibited a burst release of propofol upon reaching the BBB. 10 Synthetic block copolymers Synthetic block copolymers are amphiphilic polymers that can encapsulate hydrophobic drugs.Pluronic, poloxamer, PEG-PLGA, and PEG-PCL are examples of amphiphilic polymers that can be suspended in phosphate-buffered saline and encapsulate hydrophobic drugs. 24,26,27PEG-PCL and PEG-PLGA polymers were used to encapsulate propofol in normal saline and resulted in nanoparticle size range of 320-398 nm as measured by dynamic light scattering (DLS) with a PDI of 0.056.It is of note that DLS readings for nanoparticle size are always higher than those measured by TEM due to the hydration shell phenomenon. 46Most of the nanoparticles administered resided in the liver, lungs, and spleen while the brain had the least amount of propofol. 24,26The nanoparticles developed showed a sustained release and a half-life of 91-270 min.A comparison of biodistribution of the nano encapsulated Diprivan versus free Diprivan could have led to a better understanding of whether the developed nanoparticles were superior in passive targeting than free Diprivan.Additionally, animal studies to investigate the effects of propofol-polymer nanoformulation on LORR and paw-licking values are also needed to assess the efficacy of the proposed nanosystem.

Hydrophobic peptides
Synthetic peptides containing a hydrophobic motif selfassemble forming nanoparticles. 47Specically, peptides containing GQY amino acid motif were shown to efficiently encapsulate hydrophobic drugs including general anaesthetics such as propofol and etomidate. 37The reported entrapment efficiencies were 96.5% and 78.8% for propofol-GQY and ET-GQY, respectively.Nanoparticle size was less than 100 nm and showed promising homogeneity and stability and caused less pain upon injection.Moreover, haemolysis and cytotoxicity tests indicated safety of GQY loaded with propofol and ET for intravenous administration.GQY loaded with propofol and ET induced similar LORR timings and were deemed as efficient as free Diprivan and Forry.However, the pH reported in this study was highly acidic at 2.92, 4.23 and 6.01 for propofol, ET, and ET26 (an analogue of etomidate), respectively, which could be dangerous if administered intravenously. 37Moreover, the GQY system showed a few side effects in rats such as myoclonus.
Therefore, further investigation is needed to ensure the safety and reduce the acidity of GQY formulations loaded with propofol or ET.

Etomidate
Etomidate is a lipophilic anaesthetic drug which targets gamma-aminobutyric acid-A receptors.In addition to its rapid onset, etomidate minimally affects breathing and has hemodynamic stability making it especially suitable for cardiovascular and critically ill patients.Etomidate is 1.8-fold more potent than propofol. 37,41,48,49However, etomidate lipid emulsions have several drawbacks.It causes pain upon injection due to its poor water solubility.It inhibits 11b-hydroxylase leading to the suppression of the adrenocortical axis.It also causes postoperative nausea and vomiting. 48,49Etomidate is rapidly metabolized by hepatic esterase and its metabolites are excreted in urine.Etomidate nanoformulation and analogues have been developed aiming to retain its stability for cardiorespiratory prole whilst overcoming the side effects. 49tomidate is marketed as Etomidate-Lipuro® (B-Braun, Germany), and ForryTM (Nhwa, China) which were used in studies presented in this review.41,49 The marketed etomidates are oil-inwater emulsions consisting of soybean phospholipids and egg lecithin, and propylene glycol as a cosolvent forming etomidate fat emulsions of 168 nm particles with low PDI.41 Lipurom and Forry have a LORR onset of 8 to 12 s and a duration of 8-11 min with a half-life of 2.9-5.5 h.37,48,49 Commercial fat emulsions have mean diameters range of 200-400 nm.42 The studies investigated etomidate encapsulation focused on lipid nanoparticle formulations to reduce its side effects and improve stability of cardiorespiratory proles of patients receiving the drug.The most important ndings from these studies are summarised in Table 2.

Solid lipid nanoparticles (SLN)
The rst trials for etomidate nanocarriers were in the form of etomidate-loaded solid lipid nanoparticles with convenient loading capacity of 10% with a burst drug release of 100% release in 1 min. 43The particle mean diameter determined by DLS was 140-180 nm with a pH between 5 and 7 which gave the highest physical stability.Cholesteryl myristate (CM) was used to form nanoparticles with a smaller size of 100 nm (PDI range of 0.14-0.16)with greater stability. 50Finally, intravenous lipid emulsions were developed and exhibited a similar Nanoscale Advances Review pharmacokinetic prole to commercial etomidate as it showed low haemolysis but with low vascular irritation. 41

Poloxamer micelles
Poloxamer micelles were prepared from Pluronic F108 and Pluronic P123 micelles using thin-lm hydration and loaded with 2 mg mL −1 etomidate. 34,35Etomidate release from the poloxamer micelles was slower (68% in 6 h) than from the commercial etomidate fat emulsion (86% in 6 h). 34The average particle size measured using DLS, of poloxamer micelles prepared from Pluronic F108 and loaded with etomidate was 109 nm which was reduced to 40.5 nm, with PDI ranging from 0.250-0.296,upon adding Pluronic P123. 34,35For etimodate loaded in poloxamer micelles, the onset times of LORR in rats were 11 s, 10 s, and 8.8 s for 2.0, 2.5, and 3 mg kg −1 etomidate. 34Moreover, the duration of LORR increased in a dose-dependent manner (10, 11.8, and 13.1 min respectively).Wu et al. used soybean oil to modulate the hydrophobicity of Pluronic F108 micelles to achieve a stable encapsulation of etomidate.However, this formulation (containing 2 mg etomidate per kg) gave a shorter LORR duration of 8.5 min as compared to 9.4 min for commercial etomidate. 31tamine Ketamine (brand names Ketalar or Ketamav) is another general anaesthetic drug is used during routine surgery and also as an analgesic for pain relief.The analgesic effect is also important as ketamine is known to regulate morphine and its analogues tolerances in the central nervous system.However, it is a lipophilic drug with very limited water solubility (1 mg mL −1 of 5% dextrose or 0.9% saline) and hence low bioavailability in circulation with a half-life of 0.66-0.8h. 53,54Studies that investigated ketamine focused more on pain relief rather than loss of consciousness and used intrathecal route of administration to bypass the blood-brain barrier and avoid complications that may occur upon intravenous administration of nanoparticles.Details of studies which investigated nanoencapsulation of ketamine are presented in Table 3.

PLGA
PLGA has been thoroughly studied for ketamine's encapsulation, as it is a biodegradable polymer suitale for drug delivery.6][57][58][59][60][61] Moreover, it can be easily functionalized via apolipoprotein E (Apo-E) and vitamin E (tocopherol) for active drug targeting. 56,60Xu et al. used PLGA to encapsulate porous silica containing ketamine with larger particle size (50-60 mm) for direct intrathecal administration of ketamine towards the CNS. 59However, the study lacked in vivo assessments to demonstrate the safety of the porous silica on the caudal nerve and the efficiency of analgesia.
Targeting moieties and particle size of PLGA nanoparticles are important factors to facilitate passing the BBB.The reported PLGA-ketamine nanoparticle sizes ranged between 60-500 nm.Han et al. and Bader et al. used Shellac (a water soluble resin used in food industry) and vitamin E to improve homing of the nanoparticle towards the brain. 53,60As Han et al. demonstrated that the PEG-PLGA nanoparticles were well distributed inside the brain, liver and lungs.Similar results were reported for PEG-PLGA coated with Shellac. 53Moreover, the study investigated the haematological proles of the mice and demonstrated that they demonstrated good safety prole against polymeric materials.

Liposomes
Liposomes are commonly used for delivery of several drugs.They are made up of synthetic or natural phospholipids.These phospholipids self-assemble into small vesicles where the hydrophobic tails of the phospholipids face the inner lamellar structure, and the hydrophilic heads face the core of the vesicle and the outer part of the vesicle.Liposomes are either unilamellar (consisting of a single bilayer of phospholipids) or multilamellar.Hydrophilic drugs can be encapsulated in the core of the vesicles while hydrophobic cargo is loaded within the lamellar structure. 62Moreover, liposomes can be engineered Review Nanoscale Advances to target certain cell types by decorating their surfaces with antibodies, carbohydrates, proteins, small peptides, or small molecules. 623][64][65] One study reported preparation of multilamellar vesical liposome (MVL) and post loaded the ketamine achieving around 65.6% EE.A complete release of ketamine was achieved under 8 h, but the half-life of ketamine in vivo was 23.97 h in comparison to the commercial ketamine of 0.6 (ref.37) and 0.88 h. 54,55The liposomes were found mostly in liver, brain, and kidneys and the ketamine concentration in serum was within detectable limits even aer 5 days. 54However, analgesic effectiveness, onset and duration of LORR, and haemolysis data were not reported.

Conclusions and future perspectives
Anaesthetic drugs are used for routine surgery or for prolonged affects for terminal patients where patients develop side effects and tolerance to the administered drugs.Therefore, nanoformulation of anaesthetics was investigated to enhance safety, achieve sustained release, increase half-life time, and analgesic effectiveness upon bolus administration.However, not all nano systems work for different anaesthetics such as propofol, etomidate and ketamine.For example, loading propofol in solid lipid nanoparticles, without a surfactant, results in its crystallization.For etomidate, nano formulations (solid lipid nanoparticles and Poloxamer micelles) studied didn't improve the LORR onset or duration in rats.Also, they resulted in a burst release response rather than sustained release.In the case of ketamine, the nano formulations studied (PLGA and liposomes) showed effectiveness for 3-6 h.Therefore, further studies are needed for different formulations that prolong sustained release, enhance anaesthetic capabilities, and reduce the drug dose to reduce the side effects.
On the other hand, there were interesting formulations used that could have been further studied in terms of preclinical and clinical trial setups to ensure safety of the nano formulations; such as hydrophobic peptide of GQQQQQY, and propionylated amylose.In the case of the propionylated amylose it was The ketamine nanoparticle distributed evenly (1800 ng g −1 of tissue) in liver, brain, and kidney aer 5 days Post loading of 300 mg ketamine in 10 mL of liposome.(738 nm; 0.44) -Ketamine nanoparticles clearance was 0.49 mg mL −1 h −1 vs. free ketamine at 1.48 mg mL −1 h −1

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developed for targeting the type of phospholipids that exist only in the BBB and is considered the most plausible method for drug targeting and dose reduction.Moreover, burst release ensures fast anaesthetic response and prolongs the anaesthetic state for 30 min.More studies can be extended towards other anaesthetic drugs to test their efficiency.In the case of the hydrophobic amino acids that were used for etomidate and propofol, it was designed to reduce the use of lipophilic excipients and consequently reduce infection and induction of pain upon injection.The hydrophobic peptide also reduced the onset of LORR more than that induced by commercial products.
Investigations could be further extended towards preclinical studies to ensure safety of the nanoformulations.
Loading propofol in PEG-PLGA nanoparticles resulted in propofol biodistribution mostly in the liver, lungs, and kidneys. 26On the other hand, loading ketamine in PEG-PLGA nanoparticles showed biodistribution of the nanoparticles mainly in brain, liver, and kidneys. 53Therefore, more investigations are warranted to clarify whether the cargo (propofol, ketamine, or etomidate) could result in variable biodistribution of the PEG 5KDa -PLGA 55kDa nanoparticles.
Further studies are warranted to improve the performance and reduce side effects of general anaesthetic drugs as this would increase surgery success and pain relief while decreasing morbidities.Nanoencapsulation of anaesthetics has the potential to achieve such goals.A focus on developing nanocarriers capable of encapsulating anaesthetic drugs with high efficiency and crossing the BBB should be attempted.Additionally, designing nanoformulations which could be administered intranasally could serve as a better alternative to intrathecal administration of anaesthetics (although a wellestablished practice in surgery but causes severe complications which could outweigh its benet).Lastly, preclinical trials, using larger animals, are needed to assess effectiveness of nano formulated anaesthetics, morbidities, side effects, and pain inicted upon injection.
It is of note that nanoparticle-based systems were also developed for delivery of local anaesthetics.Porous silica nanoparticles were utilized to achieve controlled release of ropivacaine.Glycosylated chitosan encapsulated mesoporous silica nanoparticles were designed to induce prolonged analgesia in response to ultrasound irradiation. 66Additionally, hollow mesoporous organosilica nanoparticles, containing organic groups across the inorganic silica scaffold, were developed for controlled and sustained release of loaded ropivacaine for sustained local anaesthesia.The mesoporous organosilica nanoparticles can be repeatedly triggered to release the anaesthetic cargo in response to ultrasound irradiation or low pH, resulting in long-lasting analgesic effect. 67Finally, tetrodotoxin, a strong local anaesthetic, was loaded into hollow silica nanoparticles to extend its nerve blockade and lower its toxic side effects. 68

Fig. 1
Fig. 1 (a) Proposed mechanisms for crossing the blood-brain barrier (BBB).(b) Cellular composition of the BBB.(c) Methods utilized to enable materials to pass the BBB.(d) Different nanoparticles reported to have the ability to pass the BBB.Reproduced from Wu et al. with permission from [Springer-Nature], Copyright [2023].7
Fellow of Royal Society of Chemistry and African Academy of Sciences.He holds board certi-cations in Clinical Chemistry and Molecular Diagnostics and received State Excellence & Merit Prizes, Shoman Award, and Humboldt Foundation Research Award.

Table 2
Selected etomidate nanoencapsulation studies a

Table 3
Studies on nanoencapsulation of ketamine