A Focus on Fabrication, Characterization, Stability, Skin Targeting, Patent, Safety and Toxicity of Nanostructured Lipid Carrier

Background: The advanced development of lipid nanocarrier contributes a lot to the domain of therapeutic effectiveness of the drug. However the parameter such as drug loading, drug release, stability, and targeting influence much more towards the limitation of many lipid nanocarriers. The Nanostructured lipid carrier, the second generation of lipid carrier has more promising advantages over others and have tremendous targeting ability to skin for drug administration. Objective: The present review paper focus to understand the different fabrication technique, impact of lipid and surfactant on formulation effectiveness, characterization of formulation, and Crystalinity concept of lipid which have an impact on stability & drug loading. Focus on a parameter such as Transepidermal water loss , skin occlusion, and hydration which determine the ability of the carrier to target the skin. Hence the effectiveness of the drug improved. This review also focused on patents based on Nanostructured lipid carriers. Method of preparation: many methods have been adopted to prepare Nanostructured lipid carriers and among all High-pressure homogenization method is considered as best one. Review Article Sahoo et al.; JPRI, 34(17B): 49-76, 2022; Article no.JPRI.82641 50 Conclusion: Because of numerous advantages of this carrier system such as biocompatibility of lipid, high drug encapsulation, stability over others, it is considered as a major focused area for researchers. The new domain of Nanostructured lipid carrier is transdermal drug administration by targeting the skin; hence more research is focused on topical preparation. However, toxicity must have to be studied in humans. So by considering all factors one can rename it as “smart nano lipid carrier".


INTRODUCTION
In the last decade the nanoparticulate carrier imparting a promising drug delivery system for drugs. Among the nanoparticulate carrier, lipid nanoparticles carrier is the emerging carrier for recent development. As many drugs are structurally designed and well-formulated but their toxicity, low bioavailability, stability make them limited for use. Hence by choosing the route of administration along with lipid nanocarrier removes the boundary of limitation. The various lipid carriers used in the formulation are liposome, niosome, Solid Lipid Nanoparticles (SLN), Nanostructured Lipid Carriers (NLC). Among these, the NLC is now a promising carrier for researchers as it provides more advantages over other lipid carriers for drug delivery. The solid lipid nanoparticles which contain only solid lipid produce more limitations to formulation such as poor drug loading capacity( which is attributed due to lipid crystalline nature), the expulsion of drug content (because of perfect crystalline lattice formation), and stability concern of formulation over long storage [1,2]. However, NLCs are second-generation lipid carriers that consist of solid lipid and liquid lipid enhancing drug entrapment capacity and preventing leakage of the drug during storage [3,4]. Hence the current study is concerned with how NLC is a promising delivery system through the skin by studying the important parameter such as skin barrier & permeability, skin hydration & occlusion, TPEL(Trans Epidermal Water Loss), skin targeting, and stability aspect of the formulation [5,6,7]. Skin's enormous surface area makes drug ease administration and acts as a barrier for drug molecules having a molecular weight greater than 500 Da [8]. The top layer of skin called the epidermis act as a barrier that limits many drugs from their effectiveness. Hence NLC is the approach equipped with nanotechnology and lipid carriers that can make effectiveness through the skin.
NLC has particle diameter ranges from 10-1000 nm consisting of solid lipid & liquid lipid which are biocompatible. The presence of different fatty acid carbon chain in liquid lipids make NLC with a less organized crystalline structure. Hence improving loading capacity for drug accommodation. The presence of Liquid lipids is an excellent solubilizer of drugs than Solid Lipid (SLS).
The NLC produces low cytotoxicity/systemic toxicity as it is composed of physiological and biodegradable lipids. The nanosizes of lipid particles enhance drug penetration through the stratum corneum. The controlled release from this carrier is also possible due to the solid lipid matrix [9,10]. Table 1. Type of NLC model along with characteristics NLC type Characteristics References Imperfect crystal types -Nanoemulsion is formed by blending SL(solid lipid) & LL (liquid lipid) followed by cooling and highly disorder matrix formed due to crystallization process -characterized by low liquid lipid -Disordered matrix contain more space due to gap between [11] NLC type Characteristics References fatty acid chain which will accompany more drug -NLC matrix does not form a high order structure due to different chain lengths of fatty acid & other glycerol Multiple carrier types -It is Oil/fat/water system -In the solid matrix, the oil compartment distributed -High drug solubility in nanosized lipid oil compartment -A high concentration of liquid lipid is used as a drug that has poor solubility in solid lipid -Drug entrapment is more -Prolong release is achieved because of being surrounded by a solid lipid matrix -Drug leakage minimized [12,13] Amorphous types -Mixing of special lipid to form amorphous state (e.ghydroxyoctacosanyl hydroxyl stearate or isopropyl myristate) -Drug leakage minimized due to crystallization of lipid matrix [14] Fig. 1. Different types of NLC [15] 2. NLC FABRICATION

Ingredients Used for NLC
The Nanostructured lipid carrier contains major component that are solid lipid, Liquid lipid, surfactants, and water. Normally surfactants are dispersed in water and they add to the lipid mixture followed by homogenization. The ratio of SL and LL is from 70:30 to 99.9:0.1. The concentration of surfactant varies from 0.5-5% [16].

Lipids and Surfactants as Component of NLC
To formulate the NLC lipid is the primary component of the formulation. It influences a parameter such as drug encapsulation, stability, and prolonged action. The lipids used in the carrier are biodegradable and non-toxic, and physiologically acceptable. Even though many lipids are available and they have GRAS (Generally Recognized As safe) status, the choice of suitable lipid for NLC is of more concern. The characteristics such as solubility of the drug in lipid and partition coefficients are extremely vital for the selection of lipid. Most of the study reveals that the solubility of the drug in lipid influences the drug loading/encapsulation efficiency [23]. The research also reveals that drug loading, charge, and size of the particle are also affected by the degree of crystallization of lipid [24]. The melting point of lipid also has a vital role as the higher melting point of lipid leads to an increase in the viscosity of the dispersed phase which increases particle size. The other characteristics such as lipid hydrophilicity & crystal shape also influence the NLC quality. The increase in lipid amount 5-10 percent leads to an increase in particle size [25]. Hence it is highly concerning to select the suitable lipid for NLC.  [26]. The choice of surfactant is also based on the route by which the drug is administered, the effect on particle size, and HLB value. Due to crystallization during NLC formulation particle surface area increases that leading to the whole system being unstable.
Therefore the selection of surfactant becomes necessary to make the formulation stable. Another important parameter of surfactant is rHLB (required HLB) value for lipid can be calculated by dispersing in a mixture of surfactant with different HLB values followed by high-pressure homogenization to find out least particle size [27,28].
Other excipients as a component of NLC: There are other categories of excipients such as counter ion (ionic polymer and organic salts) used to minimize the problem associated with water-soluble drug encapsulation. The surface modifiers were also used to reduce the NLC formulation from phagocytic uptake by macrophages.
Various polymers like polyethylene glycol can be used to coat the lipid particle so that drug residence time can be increased in the systemic circulation. This surface modification can also improve the physical stability and drug targeting [23,29].

NLC Method of Fabrication: The
Method is Categorized into 3 Groups Namely [30] i) High energy method ii) Low energy method iii) Organic solvent-based method

Methods of NLC Preparation
High energy method based on the requirement of equipment that can produce high shear force, distortion of pressure, or the mechanism involved in particle size reduction. The low energy method does not require any specific amount of energy for the reduction of particle size. However, the solvent-based method involves the requirement of organic solvent on a mechanistic basis to the system for the reduction of particle size [30]. Among all the methods high-pressure homogenization method is the most accepted & well-reported method for the research work because of its less production time & easy scaleup process. This method is again categorized into two parts -the hot method and the cold method.

Fig. 2. Different methods used for the preparation of NLC
In the hot method, initially solid lipid has to be melted above 5-10 o c of its melting point, then liquid lipid has to be added to it & mixed for a few minutes to ensure proper mixing of it. Then surfactant solution heat at the same temperature as lipid mixture. At the same temperature, surfactant solution was added to lipid mixture followed by homogenization under high pressure (500-800 bar) to form nanoemulsion. Subsequently, the mixture allows cooling below room temperature to give NLC [31,21,32]. In the cold homogenization method, the melted hot mixture of lipid & drug is allowed to cool by using nitrogen of ice. Then the mass is ground into fine particles. The obtained macroparticle has to be dispersed in a cold aqueous solution containing surfactant/stabilizer followed by homogenization with high pressure. To get particles with average size & good polydisperse index it is necessary to use high pressure with more number cycles as compared with the hot homogenization method [33,34,35].
Ultrasonication: The cavitation mechanism used for the ultrasonication method. Initially the solid lipid and liquid lipid melted at a temperature higher than the melting point of solid lipid. The drug substance needs to add the lipid mixture. The next step involves preparing the aqueous phase of surfactant heated at the same temperature and adding drop by drop to the lipid mixture with constant stirring. The obtained emulsion is sonicated by using a probe sonicator.
Microemulsion method: Lipid mixture containing drug mixed with surfactant and co-surfactant with proper ratio to form a microemulsion. The prepared microemulsion (hot) is diluted with cold water so that breaking of microemulsion takes place which forms nanoemulsion. Solvent emulsification/Evaporation methods: This method involves the dissolution of solid lipid and liquid lipid with drugs in an organic solvent (water -immiscible). Then it is emulsified in an aqueous phase using high shear homogenization. Instead of using temperature, low pressure (40-50 bar) is used to evaporates the organic solvent. This method is suitable for the thermo labile drug as the method use low -pressure technique. However presence of residual solvent (as heat avoided) makes this method limited for use.
Membrane contractor method: The lipid mixture is placed in a pressure vessel above its MP( melting point). Under applied pressure, the lipids allow passing through the pores of the ceramic membrane to produce tiny droplets. With constant stirring, the aqueous phase allows to flow tangentially inside the ceramic membrane and remove droplets formed at the outlet. Then bring the preparation to room temperature so that lipid particle formed.

Microemulsion
The lipid-drug mixture is dispersed in the hot aqueous solution of surfactant at the same temperature to form a microemulsion. Then hot micro emulsion is poured into cold water to form nanoemulsion which will produce NLC upon recrystallization.
Scale-up process easy Dilution of the particle due to high volume of water, a high concentration of surfactant used [40,41] Phase inversion technique Under this method mixture of lipid, drug, surfactant, and water is formed by stirring & exposed to heat & cold cycle (3 cycles). Then dilute with cold water to induce shock which will produce NLC by phase inversion.

Easy process
Use of organic solvent [46,47]  -The nature and amount of drugs have a significant impact on entrapment efficiency -There is inverse relationship was observed between the amount of drug-loaded and entrapment efficiency -Lipophilic drug uniformly solubilized in LL/SL mixture and entrapped for a long period Ultracentrifugatio n & spectroscopic analysis [55,56] In vitro drug release -Factors such as liquid lipid quantity, type of solid lipid, the surfactant used, the quantity of drug and location in NLC, pH of medium affect the drug release -Release of drug from NLC controlled by diffusion of drug or erosion of matrix which depends on drug entrapped in NLC core, in the matrix or the shell.

DIFFERENT PARAMETERS OF NLC FORMULATION , ITS DESCRIPTION AND TEST METHODS
-Due to more surface area & shorter diffusion path, small particle size results in faster drug release compare with larger particle Dialysis bag method, Franz diffusion cell [57]

Parameter of NLC Formulation
Morphology: The effectiveness of NLC formulation depend on particle size and shape. The study shows that particle of NLC formulation ranges from 10-1000nm. However depending on site-specific action, the range may vary or be specific (50-300 nm for chemotherapeutic agent). The physical stability of NLC formulation depends on particle size & its distribution throughout the formulation. The parameter like entrapment efficiency, cellular uptake, the potential for a target is affected by the shape of particles present in the formulation. The analysis technique such as TEM (Transmission Electron Microscopy) and SEM (scanning Electron Microscopy) is necessary to find out the shape of the particle. Generally, NLC formulation shows the spherical particle with low surface area [31].

Surface charge:
The surface charge of particles affected by the concentration of lipid mixture and surface active agent. The term 'Zeta potential' is used to measure the surface charge. The determination of zeta potential (ZP) of a particle based on electrophoretic mobility. Higher the zeta potential value (> +30 mV) lesser the particle aggregation. Generally, the dispersion should have ZP either more than +30mV or less than -30Mv [52].

Entrapment Efficiency(EE):
Entrapment efficiency is the percentage amount drug entrapped in particle and determine the efficiency of the formulation. The lipophilic drug entrapped more as compared with hydrophilic one as the drug easily solubilized in lipid. The release rate of the drug in NLC formulation is significantly affected by EE as a high entrapment value changes the concentration gradient [55]  Solid lipid: one of the methods used to select solid lipid-based on the solubility of the drug in it. This can be performed by incremental addition of the drug to solid lipid at above its melting point until the excess of drug fails to dissolve in it. Usually, solid lipid has to be melted above 5-

LIPID MATRIX CRYSTALLINE BEHAVIOR
The crystalline behavior of the lipid matrix is to be studied as it is fundamental to optimize the formulation. The melting point depression (temperature much below its melting point) of the liquid mixture is responsible for the crystallization of lipid. The crystallization of lipid occurs only when the lipid blend of NLC cooled below its CTT (critical crystalline temperature). The crystallization of the internal structure of lipid determines the shape of the particle, amount of drug incorporation, and stability of the formulation. The characterization of Crystalinity of NLC study utmost importance as encapsulated drug undergoes polymorphic changes leads to leakage of the drug, impact on release rate and encapsulation efficiency [2,58]. There are structural changes of lipid during heating and cooling of the mixture, which lead to different polymorphic formations [59]. Therefore control of transition of the polymorphic form allows the metastable crystalline form to entrap more drugs [60] and stable polymorphic forms of nanoparticles are formed.
Depending on the cooling rate of NLC preparation and solidification of starting material, the nucleation process starts from the inner layer of lipid [61,62]. That is why depending on the preparation process and composition, the internal structure of lipid particle has various conformations like gel, liquid crystal, etc.
The study also reported melting point of the stabilizing agent can affect the lipid polymorphic form of thermodynamic stability. The melting point of stabilizer greater than 50 0 c maintains the lipid in low thermodynamic stability as compared with lipid having melting point <0 o c (which favors stable polymorphic transition) [63]. Two possible ways that the crystallization process modulation is mediated by lipid having low molecular weight. In the first way interaction between molecules of low molecular weight lipid with triglyceride molecules. On the other hand, heterogeneous nucleation process induction leads to organized of minor lipids into the micellar structure.
DSC (Differential scanning calorimetry) and Xray diffraction are the two possible methods that investigate the crystalline status.DSC gives information about the change in physical & chemical properties as a function of temperature due to heat loss or gain. This information tells about the status of lipid, crystallization, and melting of solid lipid used in NLC [23]. DSC is used to analyze the crystalline nature of lipid in a pure state and after processing (freeze-dried powder). The solid lipid & liquid lipid compatibility identified by DSC and help to analyze the polymorphic transition. The degree of Crystalinity or RI (recrystallization index) can be measured by DSC data.
Where, ΔH of NLC = melting enthalpy of 1g NLC preparation ΔH bulk = melting enthalpy of 1g bulk lipid ΔH is given in j/g & concentration given in percentage XRD is the technique that helps to determine crystal structure & various polymorphic forms and reveals compound polymorphic structural changes. In different ways, lipids may aggregate to give polymorphic forms like micelles, laminar phases. Wide range X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) give information on layer arrangement, polymorphic behavior, crystal structure. The length of long & short spacing of lattice and drug localization in it can also be studied by X-ray diffraction [64,65].

NLC STABILITY CONCERN
The long-term storage of NLC may lead to aggregation due to perikinetic-flocculation (flocculation due to Brownian motion of colloidal particles). A pearl-like network arrangement observed with NLC of highly concentrated dispersion leads to prevent collision and as a result, perikinetic flocculation can be avoided. This pearl-like network converts to fine particles once it is in contact with gastric fluid on administration [57].
As NLC formulation possesses less water in comparison with solid lipid nanoparticles, then care must be considered to avoid bacterial growth and changes in initial particle size. There are two possible ways to preserve the stability of NLC. One is to remove water content by freezedrying (by converting nanoparticles liquid dispersion to solid). The second possible way is to add a preservative to NLC preparation [66,67]. Generally, the freeze-dried nanoparticles should maintain stability by preventing changes in particle diameter, reducing reconstitution time, and maintaining the appearance while maintaining drug activity [68]. As the freezedrying process leads to aggregation of particles it is necessary to add cryoprotectant. A group of researchers (Beloqui et al ) conducted the study to know the effect of cryoprotectant on NLC formulation by taking different concentrations. of trehalose, sucrose, sorbitol (5,10,15% W/V) respectively [69,70,71].The study concluded that trehalose is effective to prevent the aggregation of particles. Another study was conducted by Varshosaz et al using microcelac, Avicel PH 102, Avicel RC 591, Mannitol, and sucrose at different concentrations and found that Avicel RC 591 at 1% concentration exhibit effective agent to prevent the increase of particle size [66]. So it needs to be attention for the formulator that the lyophilization process only does not improve stability but required adding cryoprotectant for it. Another way to prevent the instability of NLC is the use of preservatives. Obeidat et al conduct a study by using eleven different preservatives and study their influence on particle size, ZP (zeta potential) & other physical stability of NLC formulation loaded with Q10. They collected the sample at 3,6,12 month intervals (sample store at room temp.) and the result found that seven preservatives out of 11 show efficacy for the stability of NLC formulation (Hydrolite 5 was the best effective preservative) [67].
In topical NLC preparation preservative is added to maintain physical stability but preservative also causes destabilization of NLC. So preservatives are categorized into various types based on their impact on NLC preparation.
A multifactorial phenomenon is related to physical stability or the effect of destabilization. Examples of such factors are the nature of particle stabilizer, the affinity between particle surface and preservative, a preservative with stabilizer layer interaction, anchoring of stabilizer onto/into the surface, preservative ability to reduce zeta potential, surface hydrophobicity of particle [67].

Skin Barrier
Human body covers the skin as the largest organ having a surface area of approximately 2 sq.m. It serves as a permeability barrier against the transdermal absorption of many biological agents [72]. Skin acts as a major factor to determine drug delivery aspect such as permeation & drug absorption through the dermis. The skin is composed of mainly three layers such as epidermis, dermis, and lower layer of adipose tissue. The stratum corneum (SC), the outermost layer of skin is the rate-limiting barrier for the movement of various chemical substances. The coenocytes of the stratum corneum embedded in a lipid matrix have a significant role in the permeability of substance. Lipids that are present in SC are ceramide, phospholipids, sterol ester, cholesterol-3 sulfate & free fatty acid. The stratum corneum also contains sebaceous lipid (composed of triglyceride, wax ester & squalene). This organized structure of lipid is completely related to barrier properties of skin [73]. The factors which are responsible to target skin for NLC are skin permeation, skin hydration & elasticity, skin occlusion, Transepidermal water loss.

Skin Permeation
The Transepidermal pathway contains intercellular and transcellular routes as micro pathways for the transport of drugs. As two pathways involved in the transport of drugs through intercellular lipid, more research work focused on to understanding the organization of the structure and composition in the SC. The following mechanism involves drug penetration through the skin i) Lipid present in formulation mediating increased transdermal drug delivery through the appendegeal route.
ii) By skin fluidizing property lipid acts as a penetration enhancer.
iii) Direct skin-carrier drug exchange through 'collision complex transfer'.

Fig. 4. Drug penetration through the different route of the skin
Among the appendageal route, the hair follicle is the most penetration pathway for NLC. As NLC contains more lipid as a component it may exchange with skin lipid and facilitate drug penetration. However other factors responsible for drug permeation are particle size, aggregation form, the solubility of a particle in skin lipid, particle surface charge, and capacity to form a film over skin [7].

Skin Hydration
The hydration state of the stratum corneum normally ranges from 10-20%.The content of lipid and water has a significant influence on the skin frictional resistance. The presence of biocompatible lipid in NLC produces occlusive action which enhances skin hydration. Due to skin hydration, corneocyte packing is loosened and an expanded gap leads to more drug penetration [74]. As a particle of less size in NLC, the capillary channel of nanometer pores will be very smaller. Hence decrease the hydrodynamic evaporation of water [75]. When the concentration of lipid is more in formulation leads to more occlusion resulting in increased hydration. Corneometer is the instrument used to measure skin hydration. This instrument measures the conductance of the dielectric medium. The dielectric properties changes as the skin hydration level increases.

Transepidermal Water Loss (TEWL)
It is a good indicator to know the impaired barrier function of SC. It is the passive evaporation of water to the environment through the skin due to vapor pressure gradient. The increase in TEWL indicates disruption of SC and depletion of intercellular fluid [6]. When NLC is used in topical the TEWL is lesser due to skin occlusion resulting in skin hydration. The nanosizes of the particle of NLC have more surface area and improve the particle contact with the stratum corneum. The lipid particle forms a thin film over the skin and reduces the evaporation of water. The other factor responsible for TEWL is the size of the particle, amount of lipid, and presence of emollients in the formulation [76,77].

Skin occlusion:
occlusion involves hydration skin due reducing of TEWL.The presence of lipid in formulation produces film over skin leads to occlusion effect. With lipid formulation like NLC 'controlled occlusion effect' can be achieved by i) at a given lipid concentration the occlusion effect can be increased by reducing the particle size or ii) at a given particle size by increasing the lipid concentration [76]. The characteristics of lipid such as low melting point & high Crystalinity can attribute more occlusion effect.

LIST OF THE DRUGS USED TO PREPARE NLC TOPICAL FORMULATION ALONG WITH THEIR RESEARCH OUTCOMES
As the NLC remains an excellent lipid carrier, many researchers put their effort to find out the alterate route of administration. A different category of the drug such as antihypertensive, anticancer, NSAID, antidiabetic, local anesthesia, etc. is used to prepare topical NLC formulation with the aim to enhance bioavailability & avoid unwanted side effects. The lists of drugs formulated for topical use are presented in Table  6.

LIST OF DIFFERENT PATENTS BASED ON NANOSTRUCTURED LIPID CARRIER
In the era of nanotechnology, lipid carrier is attractive for formulation scientist. The researcher from academia and industry are eager to protect their invention related to NLC formulation. Every year many formulations of NLC is patented. The various patented NLC formulation is depicted in Table no 7.

SAFETY AND TOXICITY
A group of researchers (C Vario et al) conducted experimental work for the safety of NLC formulation in topical route. They used Compritol ATO, Migloyl 182 as lipid, and Tween 80 and polaxomer 188 as a surfactant for formulation. The prepared formulation was applied to the skin of the rat. It was observed that formulation remains 24 hr. in application site & no systemic absorption. Hence indicate the safety of formulation. Rahman et al carried-out research work using Zerumbone loaded NLC to know the toxicity of formulation. The oral route used for the experimental work uses mice as the animal. The formulation was composed of palm oil, Lipoid S 100, thimeosal, olive oil. The histopathological study report that the formulation does not have a toxicity effect on the kidney, liver & lungs. Bruge et al conducted research work to know the effect of various lipid carriers of NLC formulation on cytotoxicity in human dermal fibroblast using Precirol ATO 5,compritol 888 ATO, GMS, Dynasan 118,migloyl 812,softisan 100, and polaxomer 188 as ingredients for formulation. From the study, they found Compritol 888 ATO was the safest lipid as it has a neutral cytotoxic effect. V.R Salvi and P.Pawar with their research study found that because of biocompatible lipid, nonionic & biocompatible surfactant of NLC formulation without the use of organic solvent, lipid nanoparticles are non-toxic & relatively safe for ocular drug delivery [120].

CONCLUSION
NLC, a new generation of lipid carrier gaining more popularity as it has numerous advantages over others. The vigorous institutional research also progresses remarkably owing to its stability &effectiveness. The biocompatibility of lipid, high drug loading, prolonged-release, and non-use of organic solvent made the NLC more numerous areas for researchers. Among all the routes of administration skin targeting of NLC is the new domain for cosmetic research as well as topical formulation due to its occlusion and skin hydration effect. From the various methods of preparation HPH (High-pressure Homogenization) is considered as the most used method because of its scalability. The factor considered is its toxicity in humans to be evaluated. As day by day NLC formulation occupies more places in the market, we can predict its prospectiveness with more advancement in near future. Therefore by considering the above NLC can be termed as 'smart nano lipid carrier'.

CONSENT TO PARTICIPATE AND ETHICS APPROVAL
It is not applicable.

AVAILABILITY OF DATA AND MATERIAL
The data and material that support the finding of this manuscript are available on request.