MODELING AND FORMULATION OF LIDOCAINE CONTAINING LIPOSOMES

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES FACULTY OF PHARMACY

INSTITUTE of CLINICAL PHARMACY

ALEXANDER SHALAMAEV

MODELING AND FORMULATION OF LIDOCAINE

CONTAINING LIPOSOMES

Master's Thesis

Thesis Supervisor Prof. Vitalis Briedis

Kaunas ‚ 2017

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES FACULTY OF PHARMACY

INSTITUTE of CLINICAL PHARMACY

Approved by:

Pharmacy faculty dean prof. Vitalis Briedis Date:

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MODELING AND FORMULATION OF LIDOCAINE CONTAINING LIPOSOMES Master's Thesis

Thesis supervisor: Prof. dr. Vitalis Briedis Date:

Reviewer done by: Master student

Alexander Shalamaev Date Date

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SUMMARY

MODELING AND FORMULATION OF LIDOCAINE CONTAINING LIPOSOME

A. Shalamaev Master thesis/ scientific supervisor prof. dr. V. Briedis; Lithuanian University of Health sciences, Faculty of Pharmacy, department of Clinical pharmacy. Kaunas.

The aim: Biopharmaceutical assessment of liposomal lidocaine system and its in-vitro release and encapsulation efficiency.

Tasks: Validation ultra-performance liquid chromatography (UPLC) method was used for identification and quantification of Lidocaine. Establishment of liposomes formulation and quality evaluation. In-vitro release tests (IVRT) achieved by using modified diffusion cell. Methods used: Quantitative analysis of Lidocaine hydrochloride composed in formulations, in- vitro release samples and samples obtained after encapsulation efficiency testing was performed by chromatography method developed in department of Clinical pharmacy. Detection of

Lidocaine hydrochloride was achieved using ACQUITY UPLC H-Class chromatography system (Waters, USA) equipped with a Photodiode Array (PDA) detector at 230 nm. Calibration graphs were plotted according to the linear regression analysis, which gave a correlation coefficient (R2) of 0.9997. The method was tested and validated for repeatability and reproducibility according to ICH Q2 (R1) guideline “Validation of Analytical Procedures: Text and Methodology” (1) (table 1.)

Results: Optimized batches of Lidocaine containing liposome prepared with lipoid S75 of 315 mg/mL with 23.11 mg/mL tested for in-vitro releases of liposomal lidocaine showed 1 hour mean flux were 3005,67 μg/cm2 ± 721,40 while 3 hours flux were 5471,23 μg/cm2 ±1548,8. Encapsulation efficiency median was 30,8% ± 7,38%. stability test for 14 days of liposome with lidocaine showed stable pH around 6 compare to empty liposomes that showed reduction. Size were 74-81 nm over period of 14 days, while liposomal PBS fluctuated 90-110,5 nm, PDI showed PDI stability of 0,253 ± 0,054 after 14 days.

Conclusions: 1. Lipid Nanocarrier systems containing lidocaine were formulated and this is confirmed by particles size, PDI, pH data obtained from three experimental batches of optimized formulations. 2.The stability of formulated liposomal systems with lidocaine was tested for 14

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days and satisfactory stability were confirmed, further stability testing is should be performed in order to confirm long term stability of the optimized formulations.

3. Evaluation of lidocaine release from liposomal systems demonstrated relatively high flux of lidocaine if compared diffusion of lidocaine from solution through semi-permeable membrane.

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ACKNOWLEDGEMENTS

I would like to thank to Prof. dr. Vitalis Briedis for his expert advices and encouragement throughout this difficult thesis.

I’m also indebted this thesis to M.Pharm ,Ph.D. Vytis Čižinauskas which would have been impossible without the help and support through the research and hours spent in the laboratory. I acknowledge the contribution of the whole staff of Clinical Pharmacy department in the achievement of this master thesis.

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ABBREVIATIONS

UPLC - ultra-performance liquid chromatography FDA – food and drug administration

PC - phosphatidylcholines PG - phosphatidylglycerols

DRV - dried reconstituted vesicles ISCOM - immune stimulation complex LUVET large Unilamellar Vesicles o/w - oil in water

MLV - multilamellar large vesicles OLV - oligolamellar vesicles ULV - unilamellar vesicles SUV - small unilamellar vesicles LUV - large unilamellar vesicles CMC - critical micelle concentrations IV – intravenously

UV – ultraviolet

MWCO - molecular weight cutoff PDA - photodiode Array

LOD – limit of detection LOQ – limit of quantification PDI – polydispersity index PBS – phosphate buffered saline IVRT – in-vitro release test EE – encapsulation efficiency SC – stratum corneum

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GOALS and AIMS

The aim:

To develop carrier system and evaluate it by the release of Lidocaine. Goals:

1.Application of method for quantitative analysis of lidocaine.

2. Formulation carrier system with lidocaine and evaluate its quality evaluation (pH, particle size, polydispersity).

3. Examine the stability of the system.

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INTRODUCTION

Topical anesthetics intended to reduce discomfort and pain in dermatological issues (2),lidocaine encapsulated with liposome may provide topical delivery route and reduce the discomfort mentioned but a topical skin route of administration is which raises controversy questions for skin structure delivery (3) since liposomes would face with SC which can the main hurdle for drug substance to permeate. The hurdle is due to the anatomical structure which challenging the liposome transport overcome the SC. The SC barrier is mainly the unique structure of corneocytes columns which forms a bundles to prevent the diffusion through it (4). Lidocaine is most familiar used topical anesthetic. Comes from amide functional group class of anesthetics, that less risky in allergic manifesting symptoms compare ester anesthetics (2). The in-vitro release test is a manner to assess liposome quality and collection of quantitative data between different amounts of lipid in liposomal-lidocaine and would have an estimation and might use as prediction of diffusion from the SC of lidocaine liposomal formulation in the in-vivo environment, the encapsulation efficiency would show quantity of ratio between

encapsulated and un-capsulated drug portions accomplished by dialysis membrane with 10,000 Daltons (5).

Modeling and manipulation of different amounts of lipid with lidocaine in different liposomes formulation may hint and find suggestion for improved encapsulation and effective increase in solubility of the active encapsulated lidocaine through stratum corneum for further exploration.

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1. LITERATURE REVIEW

The ability of human being to visualize a small particle such as an atom caused and created a tremendous breakthrough in the technology over the years and with a mighty passion in science to understand mechanisms on the level of nano scale, this led to investigate every

technology on the spectrum of nano.

Nano scale could be very small size and according to Boisseau et al. the scale is about 100 nm or less (6), many cells, proteins and human components are about this size range.

Reference to the U.S drug and food administration (FDA) which also defines what considered as nanotechnology confirms by stating material from 1 nm to 100 nm can manifest different

‘chemical or physical properties, or biological effects compared to larger-scale counterparts’ (7) Nanotechnology is a science of engineering, and technology which leads to the nano scale range (8), a study by Filipponi shows that Nanotechnologies are based on the manipulation, control and integration of atoms and molecules to form materials ‘…’ (p. 23) (9).

The integration of atoms onto such a small particle size requires new approach of thinking when dealing with human body components.

New nanotechnologies are developing right before one’s eyes but to deal with such small scale requires deep research and fieldwork before releasing new technology especially in the field of Nanomedicine and pharmaceutical administration.

1.1. Nanomedicine development

Rapidly, Nanomedicine initiated from nanotechnology or as stated Boisseau et al. ‘There is no Nanomedicine, there is nanotechnology in medicine’ (6) which begun develop through the years with the knowledge so that scale at which most of the biology occurs with the awareness that Nanomedicine has the potential to develop more opportunities for development of better healing options, a recent study approve that human body components works at the mentioned range above (10).

Boisseau et al. has drawn attention to the fact that Nano medicine engages at same frame scale as about 100 nm (6), which confirm that nano medicine field going to a smaller and smaller

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the answer is to be found in the fact of physical law apply in case of nano scale materials such as the increment in surface area over the volume area is indirect proportion to molecule size. Furthermore, Nano medicine provides a new and better cure and diagnostics for a large scope of pathologies (11),might allow health care providers better understand the pathological cause of certain diseases.

Nano scale research and development clinched by the pharmaceutical area of science and by many more branches such as (6):

• Delivery of pharmaceuticals

• in vitro, on vivo and in vivo diagnostics, including imaging. • regenerative medicine.

• implanted devices.

The fusion of all Nano medicine branches might bring to the science lots of solutions in the medicine sphere, and the point of convergence in pharmaceutical area is the delivery of drugs behavior which only developing in recent years may sway for the benefit of therapeutics with better delivery and show progress in clinical researches, especially when in-vitro and in-vitro involved with medicine and medicine of regeneration is takes place too.

1.2 Delivery essence

Pointing out to the delivery of pharmaceutical, that brought to science world the Nano pharmaceutical term which mainly deals with drug delivery, and drug formulations at the nano size.

As a manufacturing step each drug preparation passes through the formulation course before application which means how drug will be delivered, a well-known ways of drug delivery is enteral, parental, inhalations, topical and many more, there are several systems of drug delivery such as dendrimers, nanocapsules, liposomes, solid lipid-based nanoparticles, protein conjugates, micro - and nanoemulsions, inorganic nanoparticles, carbon nanotubes (12), as common base to some of these systems is the colloidal system that many attempts in the last 40 years have been under investigation to seek for colloidal nano scale particles for drug delivery (13).

main goal of drug delivery according to Hafner et al. ‘improve drug solubility, extend drug half-life, improve a drug’s therapeutic index’ (14), worth to mention that colloidal nano size particles

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for drug delivery share the same properties such as spherical shape with large capability of drug loading (15). Already for a five decades the science knows these spherical are able load and filled with therapeutic agents materials with ability to deliver therapeutically agents onto specific location in the human tissue or body (8). Example of successful story and achievement of the Doxil® drug which is considered as the first approved nano scale drug by the U.S food and drug administration (FDA) at the year of 1995 (13). The revolution continued, the year after the FDA approved one more nanopharmaceutical drug known as DaunoXome® as the active substance is the Daunorubicin indicated to HIV-related Kaposi’s sarcoma (13),by 2011 there are 22 approved nano drugs by the FDA (6).

To sum up, harsh diseases are getting treated as an example the HIV-related Kaposi’s cured by nano pharmaceutical just may hint what’s the future holds for breakthrough of new indications and higher rate of treatment with lower percentage of morbidity can done by nano drugs which not surprisingly research begun previous decades and new area of development of nano drugs begun, this symbolize the willing of pharmaceuticals firms to look for more nano drugs in order to improve the therapeutically drug delivery as with the successful achievements of Doxil® and DaunoXome® and many more, and seems like science community are not far from the step to cure or the least to extend the patient life’s.

1.2.1 Targeted delivery problem

The outermost layer of the epidermis as known as the stratum corneum (picture 2,page 29) consist of corneocytes like bricks and mortar shape in a lipid matrix which consist of cholesterols, ceramides and some free fatty acids, that arranged in very ordered pattern with repeated space between each other makes it as the main barrier for mobilizing of substances to penetrate into the skin (16),furthermore, the movement of substances into skin can be expressed by diffusion by intracellular route and most of compounds penetrate through the lipid

membranes (16).

Evidence for penetration can be found easily in the literature and ease of penetration through skin and stratum corneum can be pointed out as specific targeted delivery with many advantages. Stratum corneum has a very special lipid configuration the mentioned above ceramides with long

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strings, cholesterol and free fatty acids that rules distinct nature for the lipid phase in analogy to other biological membranes. i.e. lucid lipid are highly prevalent but a smaller division of these lipids creates a liquid state and for that liposomes adjustments is the is the design in order to increase the facility of the doubled layers and fusion options to penetrate crosswise the lipid matrix (16). Due to that reason mentioned skin site of application is barrier but nevertheless liposomes are attractive approach that intended to overcome through this hurdle and diffuse through the though skin barrier and could be a tempting filed of research in the area of skin targeted delivery.

1.3. Nanoparticles

The main goal of nanoparticles is increase bioavailability, biocompatibility, better cure efficacy, stability and solubility of drugs, and to avoid in some extent their adverse reactions (17).

These nano particles could be classified as matrix type or encapsulated particles, thus either dispersed identically or in the polymeric matrix or wrapped inside (18) ,as well as these nano carriers which able to carry the drug on made of which fits for human organisms or either by biodegradable materials (19).

Some of the nano carriers has a spontaneously self-aggregation feature (13).

A broader classification of these self-aggregates can be classified into conventional, long circulating and actively targeted system. The conventional carriers could be further divided into liposomes and nano particles (20).

Potentially and practically nano scale technologies are already emerged in many areas of research, as mentioned above with proven nano drugs are on the market. Assuming that nanotechnology provide us a better understanding of the diseases and with combination of the drug delivery exploration can produce magnificent results in areas of pharmaceutical trials, as well as drug delivery, the evidence seems to indicate that especially when bioavailability and biocompatibility would significantly increase along with stability we can predict a better result in clinical errors when the therapeutic index will enlarge as stated by Hafner et al. previously.

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2. CONVENTIONAL CARRIERS

Many nano scale carriers have been drawn the attention of drug delivery system with proven and solid evidence of marketed drugs, a large sector of biodegradable organic material such as phospholipids that is the elementary unit for liposomes framework and formulation (21). Phospholipid acknowledged as the fundamental part of the biological membrane so assuming that the proclivity of phospholipids to form liposomes which are self-aggregated in the aqueous environment is well-known (22), and reasonably occurring naturally.

This leads to the understanding that conventional carriers both liposomes and nano particles has similar properties which can be differentiate by composition, size, size distribution, surface charge, number of phospholipids bilayers and differ from nano particles in the matrix compactness which is a unique feature of nano particles (23).

Speaking of liposomes, meaning originally comes from the Greek language and means fat body when lipo is (“fat”) and soma is (“body”),liposomes described well already in the previous century (24),and yet they are in huge exploration, as a broad term they can be featured with an aqueous media in the interior lacuna or a few of them which enclosed with phospholipids bilayers (13).

To sum up the basic idea behind nano carriers which can be manipulated into variety optional manipulation of size, composition, surface charge and many more physiochemical features would be more easily to aim and direct the nanocarriers into certain tissue of biological membrane especially where acknowledgment of proven nano carriers are in research since the last century and most likely scientists has expectation from previous works done in the field that’s could be preferable system to work with.

2.1.The trailblazer of liposomes

Lipid vesicles which is not mistakenly called liposomes. Brief history reveals the pioneers of liposomes area were Alec Douglas Bangham and his colleagues in the early 1960’s who noticed that some blur of egg lecithin reacted with water to form quite intricate structures (25). Product analysis preformed with electronmicroscopy exhibiting that a multitude of vesicles were formed immediately and first called ‘smectic mesophases’ (25).

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As Bangham dubbed them as ‘Bangosomes’ firstly and only later liposomes termed by him, but first who could realize that they can enmesh were Gregory Gregoriadis (24).

On the one hand lipid vesicle was made of phospholipids and considered as vital for biological activity, this basis was known for pioneers at that era of time, but on the other hand classification of various wasn’t known since that was only the beginning of liposomal aeon.

Since that period of time lots of changes have been occurred and nano carriers developed tremendously. Picture 1 depicted an illustrative representation of liposome, in green color the phospholipid bilayer is shown and in between the hydrophilic and hydrophobic in red and blue respectively.

The evidence seems to indicate that experience in the field of nano carriers is there for more than 50 years and yet feels that the full potential from nano carriers isn’t achieved yet, and plenty of options are yet to be discovered.

Picture 1 - Simplified sketch of liposome (26)

2.2.Physiochemical aspects

Spherical shape of self-aggregation is composed clearly from some molecules so these carriers obviously composed from lipids, and more specifically phospholipids used as building block in the case of liposomes.

Phospholipid is molecule that is incorporate phosphorus element with polar head and non-polar tail (22), that share same physiological properties as human membranes, phospholipids

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originated from the following two origins, natural and synthetic ones (22), first and most common phosphatidylcholines (PC), be in the possession of naturally lipid class, and

phosphatidylglycerols (PG), correlate to the anionic lipid class (synthetic), are mainly used in drug delivery products.

Self-assemblage is unique to phospholipids in aqueous into hexagonal conformation for example liposomes or micelles (22).

Important feature of characterization of liposome is the zeta potential which is the electrical charge of liposomal surfaces that can determine stability under biological environment (21). And maybe would be right to say the outcome of the liposome and on this basis it may be inferred that the physical phenomena of zeta potential have to be taken into consideration in order to predict the consequence of the liposome since size is might indicate the permeability of the liposome can determine what route eventually will be formulated, and charge can be helpful tool to decide whether the liposome is suitable for the needs.

3. LIPID VESICLES TYPES AND CLASSIFICATION

3.1.Variety of species

There are many types of lipid vesicles or vesicular colloidal spheres as well as the

composition of each type is differed from each other. Several studies have classified liposomes by different categories ,many publications about liposomes classifications and the types of them are exist and written but briefly important to know there are many types of based and categorized on several parameters such as based on structure, composition, and preparation (25) and the following types is the main five types (5):

• conventional liposomes, main focus given onto conventional. • pH-sensitive liposomes – subdivided into four more subgroups. • cationic liposomes

• immunoliposomes

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on the contrary another publications specifies the various types of the nano lipid vesicles into long list of more than nineteen different types of them, overview of them will sum up that they different in function (25):

1. Archeosomes – As hinted from the name itself contain of archebacteria lipids the distinguished from the prokaryotic and eukaryotic.

2. Cochleates – Special types of liposomes which suspended in an aqueous media in more than one polymeric solution phase, usually charged with the cationic ions of calcium either magnesium to for a precipitate.

3. Dendrosomes – Family of lipid vesicle with many advantages which is stable and cheap to apply and use in the field of vehicle for gene therapy.

4. Dried reconstituted vesicles (DRV) – Single unilamellar vesicle with different lipid mixtures of them, the method of preparation is focusing on creation of small and hollowed vesicles. 5. Ethosomes – Mainly used in the territory of pharmaceutical skin delivery and composed from

soy phosphatidylcholine and ethanol in certain percentage to present multi layers of vesicle. 6. Immunoliposomes – Contains certain antibodies fragments such as Fab’s (fragment antigen

binding) or peptides on top of it there is a doubled layer which formed for in-vivo or in-vitro use.

7. Immunosomes – Small size vesicles, unambiguous name and used for immunological fragment or structures for the establishment of in-vivo or in-vitro utilization.

8. Immune stimulation complex (ISCOM) – Composed of saponin mixture are about 40 nm. Encompass some antigens.

9. Lipoplexes – Positively charged ions NDA lipid compound used as carrier for cell transfection, with certain toxic effect.

10. Large Unilamellar Vesicles – Abbreviated as LUVET, also known as Novasomes, prepared by extrusion method.

11. Niosomes – Small unilamellar vesicles prepared by unionized surfactants, also known as Novasomes and almost similar stability as Archeosomes.

12. pH sensitive liposomes – This class divided into four different species: • polymeric lipids – Functions as neutral pH stabilizer

• liposomes comprise lipid by-products with higher permeability

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• pH-titratable destabilize membranous at acidic pH

13. Polymerized liposomes - varies between 35 up to 135 nm with Phosphatidyl choline vesicles owing one or two methacrylate units for single compound.

14. Proliposomes – anhydrous form with flowing element which instantly establish liposomal dispersion in a watery environment.

15. Reverse-phase evaporation vesicles – Known and abbreviated as REV, created by vaporization in system of o/w (oil in water) emulsions to bear a unilamellar liposomes. 16. Stealth liposomes – They are synthetic liposomes with polyethylene glycol lamination which

is hydrophilic polymer that enhance stability and half-lives in circulation. The cover can avoid proteins to adsorb and avoid elimination from the human body.

17. Temperature-sensitive liposomes – They are ensuring the delivery of drugs into definite location with aspect of attraction to the cell surface when this attraction is controlled with temperature dependence.

18. Transferosomes – Contains phosphatidylcholine and cholate with drastic anamorphosis trait and has a skin delivery ability.

19. Virosomes – Small and unilamellar encapsulated with influenza hemagglutinin, can enhance some immune response.

It is clear therefore that the mentioned above types list contains almost all the known Nano carriers that literature has to offer with many functions in the field of medicine, or

pharmaceutical, the wide range of functions of the listing above offers in-vitro or in-vivo

predications which can help and guide science for better treatments options due to that the most important is knowing with earlier knowledge what kind of nano carrier required in order to maximize the exploration, in the case of single unilamellar vesicles most critical to look for route of application due to permeability issues of the content in the nano carrier inside and since lipid vesicles are coming in different sizes allow the scientist plentiful options such as that can be seen in the research done with Transferosomes to enhance transdermal delivery of Sertraline (27) to vary with vesicles and investigate them for the many purposes especially when literature is supplies the framework for preparation methods in a reproducible routine, yet it’s necessary to be sure with the nano carrier chosen, so personalized medicine suddenly not sounds like impossible.

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3.2.Classification and typology

As mentioned above furthermore categorization ways exist to classify, here sizes are nanometers scale. according the work done by author Akbarzadeh et al. which did the division according to size (28).

• Multilamellar large vesicles (MLV) – greater than 500 nm • Oligolamellar vesicles (OLV) – between 100 nm to 1000 nm • Unilamellar vesicles (ULV) – all size ranges

• Small unilamellar vesicles (SUV) and Medium sized unilamellar vesicles (MUV) – 20-100 nm. • Large unilamellar vesicles (LUV) – above 100 nm

• Giant unilamellar vesicles and Multivesicular vesicles – more than 1000 nm

Based on methods of preparation, Garg et al. have considered techniques of preparation as necessary division (29) , as given here the list easily and early conclusion my come up that there is not only one way to prepare these lipid spherical shape encapsulated with substance in order to apply for several route of administration. the methods brought here are not identically to each other, as example Niosomes are considered as SUV and can be used as topically or as inhaled treatment for pulmonary treatment conditions (30).

• MLV - Thin film hydration (hand shaking), rehydration • ULV – Thin film hydration (no shaking)

• SUV – Micro fluidization, Ethanol injection, Freezing/thawing • LUV – Reverse phase evaporation, Detergent dialysis.

As referred from the paragraph above many techniques exist might indicate that nano carriers are one step closer to create the ideal term of personalized medicine since every techniques and class of nano carriers can be intended into specific tissue seems like that pharmaceutical are in progress to cause medical treatments are could be asked by personal demand and as evidence 12 drugs approved by FDA from 1995 TO 2012 (31). The only question is in doubt left here, does it can pass the level of idea only and get into the clinical phases.

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4. TECHNIUQES OF NANO PREPARATION

Although liposomes are nano scaled, the preparation is not performed by one method and varieties of methods are exist and choosing the appropriate one might be the rate determining step in order to achieve required size.

Liposomes can trap the drug in two ways and as according to Akbarzadeh ‘active loading techniques and passive loading techniques’ (28). Passive methods are demonstrated in Figure 1 that exhibits also the passive and the active techniques, certainly the passive one is split up into three different branches and mainly the mechanical is the broader one, the sonication method is divided into to two methods of preparation and distinguishable by minor properties.

Main difference between the passive and active is the passive will enter through the passive diffusion way due to the fact skin has many sponge like blood capillaries that are passable to particles of 100 nm (31). Active loading techniques involves devices which increase the permeability of the drug into the skin in order to have a better penetration, there are several techniques (3),which is not the main core here.

As a general rule classical ways that includes passive and active has a four steps that involves in the production of them (28) :

1.Drying the lipids from organic solvent (ether, diethyl ether) 2. Dispersing the lipid in aqueous media.

3. Purifying the liposomes.

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Figure 1-Methods of nano sized vesicles preparation (25)

4.1.The passive load

1. The detergent removal technique is elimination of non-encapsulated substances (28), briefly speaking detergents reaches into critical micelle concentrations (CMC) and this intended to dissolve lipids. As the detergent is isolated, micelles getting more and more better-off in phospholipid and finally blend to form large unilamellar vesicles. (28)

2. Solvent dispersion method is performed under pressure of vacuum when a solution with lipid is dissolved in organic compound and the encapsulation is performed under temperature of 55-65 C° (28).

3. Mechanical dispersion method is the broadest group to cover with many methods which involves the use of several techniques, and basically, they are homogenization Techniques. Homogenization techniques intended for breaking up the cells which mean useful for size reduction and number of bilayers liposomes (32)

i. Lipid film hydration is known as the Bangham method , that requires the hydration of lipids by rotation movements (5).

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unstable materials, resulted in liposomes are larger than small unilamellar vesicles but rather multilamellar (28)

iii. Freeze and thaw method based on small unilamellar vesicles that quickly frozen and defrost gradually, afterwards a brief sonication and aggregation occur to level of large unilamellar vesicles. (28).

iv. Micro emulsification - Method provides liposomes with a high aqueous volume and the lipid ratio and ability to encapsulate a large proportion of the aqueous material given (28).

v. Membrane extrusion - Conversion of multilamellar vesicles (MLV) into small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV). In the membrane extrusion process of a suspended MLVs is ejected certain times through homogenous cylindrical pores of a track engraved polycarbonate mem-brane to have a smaller vesicles as result (33).

vi. Dried reconstituted vesicles – First of all the dispersion is freeze then only drying taking place the dispersion of unfilled small unilamellar vesicles and then rehydrating it with substance containing aqueous (29).

Thus it could be concluded that passive load techniques methods holds a broader spectrum of preparations in mechanical way instead of the two other ones that’s most likely due to

homogenization is required and sonication method can provide it easily with the option of choosing the amplitude and manipulation of size variation, but easily one can get lost in the selection of suitable method, in a personal note have to add that sonication method were easy and convenient method due to simplicity of usage the only caution have to be taken is heat labile vials have to be carried and avoidance of probe touching with the side walls of glass to dodge of glass vial breaking.

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4.1.1. Ultrasonic as the disruption method

First of all, sounds wave which are above the audible sound are named as ultrasonic (>20KHz).

Sonication or ultrasound technique not new into science world and used for several decades, more interestingly is how this method can yield a sufficient vesicle in point of view of required size and many more physiochemical aspects.

The sonication or more precisely ultrasonic technique considered by the list above as the seventh method but the main one for disruption from multi into unilamellar vesicles large unilamellar (33). The method uses ultrasound energy that distribute engage low frequencies with an unfocused and not entirely controllable energy output (34).

Since this method is properly intended for disruption some publication would name it as

downsizing technique due to the reason we are lowering the size of the vesicle formed from large size into the spectrum of defined sizes. To decrease in size of the liposome required by the use of sonication method and likely that without this step the lipids are not uniformed in their sizes (33).Sonication method is divided into to two subdivided methods of preparation and

distinguishable by minor properties but yet with the ultrasound technique. The probe and the bath sonication (34).

These methods use sonication with a small difference in probe sonication the tip of a device is directly immersed in vesicle solution vial. Very high energy input invested into solution. It may result in hot vial due to the invested input, accordingly, the vial has to be dipped into a very cold as ice bath, in the probe sonication style, the tip made of titanium and may contaminate and stain the solution by peeling of the titanium probe (28).

In the contrary, the bath is the sounds like more easily treat the temperature problem mentioned in the tip but it works in the manner that liposomal solution that in the vial is fixed into a bath sonicator and temperature regulation is made manually. The solution is covered in a sterile separate vial, differently to probe method (28).

It must therefore be recognized that downsizing is required form for reduction of vesicles but yet no uniformity in size achievable which can cause in different polydispersity index and not uniform formulation this obstacle have to be checked before, on the other hand it can’t be taken

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from the method that is already proven in previous so more convenient ways maybe exist but that one is makes all the work is required although the pros and cons it has to offer.

5. ADVANTAGE AND DISADVANTAGES

5.1.Drawbacks

Liposomes functions and variety of usefulness areas that liposomes are taking part as carrier or drug approved for therapeutically use, necessary to discuss benefits and the

disadvantages of them to imagine how liposomes can change the future approach for drug encapsulation approach for route of administrations with focus on skin delivery, benefits and disadvantages are always aspects for considerations ,advantages that drug encapsulation would offer (28):

• Higher competence for solubility for lipophilic and amphipathic molecules

• Through the passive diffusion mechanism, the direction of vesicle into the immune system cell is addressed.

• Either systemic or local administration of liposomes could be maintained by slow released and the control is achieved.

• Ionized and molecules with charge has a better transport with better permeation into tissues.

5.2.Advantages and benefits

Clearly and most probably these advantages have many interest to offer for the therapeutically skin delivery. To get into the core of benefits from the liposomes which has several options to suggest in favor with their functionality (28).

• Higher competence for the pharmacological therapeutic index

• Drug molecule could gain more cohesion through the entrapment in it. • Safety aspect with biological bio-adaptable for human physiology • Reduction the lethal effect of the entrapped agent

• Liposomes help reduce the exposure of sensitive tissues to toxic drugs

• Affinity to bind precisely to tissue ligands to accomplish effective active targeting

With the leverage of the liposomes with the list above, easy to list the imperfections and drawbacks of liposomes (28):

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• Protection against oxidation and hydrolysis reaction of phospholipids can occur. • The escape and breakage of the entrapped drug

• Production cost is high due to the device required • Stability tests necessary

The information above provides the schematic hurdles that will be hard to overcome and due to that taking the advantage of previous works and investigations has to be taken into consideration in order to minimize hedgerow.

This consideration together with physiochemical aspects is important especially the permeation through the skin and diffusion that has to be taken into examination, worth to conclude that difficulties to implant systematic sonication energy output in large extant to larger volume of substance, always the exposure of degradation reaction due to redox and degradation of

phospholipid is exist, and metal sloughing off from the tip probe is well mentioned in previous works (35).

The evidence seems to be strong that despite all the difficulties above already marketed liposomal drugs are approved such as Visudyne® drug which contains Benzoporphyrin for treatment of predominantly classic subfoveal choroidal neovascularization due to age, pathologic myopia, or presumed ocular histoplasmosis (36) and the another one is Marqibo® that got the approval in 2012 for the indication of acute lymphoid leukemia, Philadelphia chromosome-negative, relapsed or progressed (IV) the size is around 100 nm with Vincristine sulfate

entrapped in sphingomyelin/cholesterol (13),all of mentioned are injections and marketed for the beneficial of patients. The more interesting question does topical route is part of the future plans for pharmaceuticals firms to produce reliable liposomes that doesn’t require the dependence of injection and reduction of injury risk at the site of application is a point to explore.

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6. SKIN AS HUMAN FENCE

Human skin is an organ by itself and well known as pharmacological route of

administration for gels, creams, ointments, and surely for many more medicaments as local and systematically route for delivery. Human skin is obviously the first fence that molecules or particles meets and functions as natural border for the avoidance of foreigner and unwanted micro-organisms entrance. Skin is one of main areas that topical substances can be absorbed into it (37). According to Prow et al. states that the skin has provided preventive structure which give it physical, immunological, metabolic and UV-protective fence to grant it to prohibit invasion by any toxic effect (38).

As a side note skin treated as largest organ of the body, estimated for about 15% of the total human adult body weight (39), not surprisingly the first layer which known as stratum corneum (SC) is the first barrier for many unwanted particles.

6.1.The skin anatomy and complexity of entrance

The skin is composed of three layers: The Epidermis, the Dermis, and subcutaneous tissue the first called the Epidermis, contains of a definite structure of Keratinocytes cells, that synthesize Keratin, protein with a protective aspect. The second and middle one layer, called Dermis, is essentially composed of the Collagen, this layer is Dermis in contact on the subcutaneous tissue, of which consist small convex shaped of fat cells termed lipocytes. Thickness of layers is varying and dependable on the area on the human body.

Issue with that kind of administration and answer in generally that skin delivery could be convenient and not requires the patients to face with solid dosage medications and the issue that accompanies to that.

On the other hand, drug delivery into the epidermis and dermis without problems and modification has met issues in the past with little success (38). Over the last year and more recently particle construction formulation in the pharmaceutical society science acquired great acceptance of nanoparticle–skin interactions will easily point to significant clinically

advancements in topical delivery research (38). Thus it could be concluded that penetration of any substance into skin isn’t easy as thought and early thoughts have to be given before placing

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nanocarrier to work with especially when speaking of the skin which composed of several layers that can accumulate most likely different concentration in every different dermis.

6.1.1. Stratum corneum as a skin shield

Smooth, having no nucleus, stratum corneum are tightly arranged in the extracellular lipid matrix which is organized in manner of bilayers a special termed stated by Michaels et al. in 1975 termed ‘bricks and mortar’ frame-up. The corneocytes are adhered together by corneo-desmosomes that aid to form a stiffer exterior layer by keeping cellular configuration (38). To cross the Stratum Corneum passive diffusion has to be done and based on the

dual-compartment bricks and mortar model (picture 2) of the stratum corneum, disrupted external help will happen with possible three ways (38):

1.transcellular 2.intercellular

3. appendageal routes

Most penetrable path is the intercellular route is governed, according to Prow et al. which states that small molecules are able to move freely within the inter-cellular spaces and diffusion rates are governed largely by their lipophilicity (38).

One more issue to deal with is the danger of adverse drug reaction is always in questions due to reason systemic and local delivery might reach the blood circulation and this even makes it even harder to find an answer for that.

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Picture 2. Cross section of skin morphology and brick and mortar model (2)

To sum up stratum corneum represents the main problem for permeation of topical delivery and might be defines as the rate limiting step and finding solution for that might be no an easy topic and the main debate here will be the answer how to improve efficacy of skin penetration with certain molecules.

7. LOCAL ANESTHETICS AND NANO-CARRIERS

Topical anesthesia is a useful method to decrease pain at the required skin site and considered as well to reduce malaise at the site of skin before to application during dermatologic treatments. Certain local anesthetics compounds for skin anesthesia are the following (16):

• benzocaine (aminoester) • prilocaine

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Three above are commonly known in several formulations such as gels, ointments, creams (benzocaine and eutectic mixture of lidocaine and prilocaine) or as a biological adherent (lidocaine) with various designs (carriers and no medically active substances) (16). Speaking of Lidocaine was were synthesized for the first time by Nils Löfgren in previous century at the year of 1943 and topical delivery of Lidocaine used during the years with safe use but with exposure to toxic and allergic concerns (2).

Liposomes loaded with drug agents has already proven the diffuse without any resistance through the passive way and pile up at the site where the permeation is increased just as precondition carriers has to be less than 200 nm (24).

For route of topical administration, nano lipid vesicles as liposomes with various sizes delivering the wrapped drug to layers of skin, with augmented penetration into deeper layers of the skin demonstrated by liposomes of smaller sizes such as mentioned above (36).

To sum up Lidocaine inserted into liposome in order to penetrate the skin mainly the stratum corneum and the justification have to be explained due to instability of lidocaine itself to permeate, obviously is a problem of delivery of substance such as that which is lipophilic and soluble in lipids and the wanted result is to invade it and not let it dissolved on the outermost layer. Liposomes can help penetrate in through the skin layer but in second thought what could be considered as successful, the permeation or the concentration penetrated into skin when the goal is decrease pain and pain is relatively ratio in different individuals, this section could be the next level of research.

7.1.Evaluation of lidocaine concentration

Already we have seen that liposomal drugs are penetrating the skin and especially the stratum corneum, but how to evaluate the amount or quantity that penetrated already, this question is most probably the most important to know since estimation of concentration has to be accurately in order to achieve therapeutic progress.

Many studies with different methods have been performed in the past in order to estimate concentration of lidocaine in the skin examples begins with suction blister method preformed back in 1968 as well as punch and shave in 1993 through autoradiography in 1978 as well

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heating in 1990, all these methods unsuccessfully got into conclusion that difficulties in measuring proper concentration (40).

7.1.1. In-vitro release test

Method of in-vitro release (IVRT) test through membrane dialysis is the measurement of drug release through diffusion process this method is the most common one, the IVRT is a tool to give some indication regarding the behavior of agents in in-vivo milieu (41). Although this tool officially entered to regulation in the American pharmacopeia for IVRT only in 1970 and yet there is not accepted pharmacopeial test for nanovesicles (41). This method can conclude the amount of accumulated donor in the acceptor and sampling at variable times. certain restrictions alters the drug release in the dialysis methods include disturbance settings, proportion of donor and acceptor cell volumes, and molecular weight cutoff (MWCO) of the dialysis membrane, very important preference for an optimal dialysis membrane with the better possible pore size to grant free drug diffusion, and yet the pore size must be small enough so that the nanovesicles won’t be able to pass by (41).

The evidence seems to indicate that in-vitro release test is not acknowledged in many publications and yet does in the American pharmacopeia could hint that this test is may be a good prediction for the in in-vivo course of drug substance, but since there are many questions behind this method such as the reaction of the donor wall device with the medium examined (41), as a conclusion this method can point on in-vivo significance prediction but seems not enough, and can be in doubt for future research. Another point is the membrane which is

surrounds the acceptor have to be checked for various types in order to have and compare results of the same diffusion rate.

8. MATERIALS AND METHODS

8.1.Materials

S75 phosphatidylcholine soybean phospholipids with 70% phosphatidylcholine were purchased from Lipoid GmbH (Ludwishafen, Germany).

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Lidocaine HCl purchased from MOEHS CATALANA S.L, Poligono Rubi Sur – Cesar Martinell Brunet, n° 12A,08191, Barcelona, Spain.

Phosphate buffered saline purchased from Gibco by life technologies™, UK.

Membranes made of Cuprohan sheets with MWCO of 10,000 Daltons, DIMESIONS 500 x 500 mm, purchased from medical membranes Ltd 239 Liverpool Road, London N1 1LX.

ACQUITY UPLC H-Class chromatography system, Waters Acquity UPLC System, Waters, MA, USA.

Stirrer used were WiseStir model MSH-20D by Wisd laboratory instruments, Dublin, Ireland Sonicator model HD 2200 purchased from BANDELIN electronic GmbH & Co. KG

Heinrichstraße 3 – 4 • D-12207 Berlin.

Zetasizer nano series purchased from Malvern instruments, Enigma Business Park Grovewood Road, Malvern, WR14 1XZ, United Kingdom.

pH meter: pH meter model 766 with suitable probe of Knick SE 100 N, Knick Elektronische Meßgeräte GmbH & Co, Germany.

All materials were used as purchased

8.2. methods

8.2.1. Quantitative analysis of lidocaine hydrochloride content

Quantitative analysis of lidocaine hydrochloride content in the formulations, in-vitro release samples and samples obtained after encapsulation efficiency testing was performed by chromatography method developed in department of Clinical pharmacy. Detection of lidocaine hydrochloride was achieved using ACQUITY UPLC H-Class chromatography system (Waters, USA) equipped with a Photodiode Array (PDA) detector at 230 nm. The column was a Acquity UPLC BEH C18 (130Å, 1.7 µm, 2.1 mm X 50 mm, Waters). The mobile phase was delivered in a linear elution gradient from 15 % to 25 % of solvent A (Acetonitrile) in B (0.1% (v/v)

Trrifluoroacetic acid in ultrapure water) for 3 min; the injection volume was 1 µL, flow rate was 0.65 mL/min, and the column temperature was 30 °C. Elution time of lidocaine HCl was 1.142 min. The limit of detection (LOD) was 0.993 µg/ml and limit of quantification (LOQ) was 3.992 µg/ml. A standard calibration curve (peak area of lidocaine HCl versus known drug

concentration) was built up by using standard solutions (4 – 75.58 µg/ml). Calibration graphs were plotted according to the linear regression analysis, which gave a correlation coefficient (R2)

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of 0.9997. The method was tested and validated for repeatability and reproducibility according to ICH Q2 (R1) guideline “Validation of Analytical Procedures: Text and Methodology” (1) (table 1.)

Table 1. Validation parameters of chromatography method for lidocaine hydrochloride

Compound Repeatability Reproducibility

Accuracy, % Precision, % Accuracy, % Precision, % Lidocaine HCl 95,6 – 102,8 0,49 – 1,33 96,2 – 99,1 3,01 – 4,72

8.2.2. Preparation of liposomes with Lidocaine

PBS solution with Lidocaine freshly prepared with concentration of 23.11 mg/ml, a portion of PBS-Lidocaine poured on to PC S75 and stirred for 4 h at room temperature condition. The preparation kept in storage condition and sonicated the day after in a manner of 30 sec. on and 30 sec. off with 0 cycles and amplitude varied from 26%-30% of maximum. IVRT and

encapsulation efficiency (EE) preformed the day after only.

8.2.3. Characterization of liposomes 8.2.3.1.Size and ζ- potential.

The mean diameter (z-average) and ζ-potential (zeta) of the liposome were measured by a laser-scattering technique (Nano ZS90, Malvern, Worcestershire, UK). The formulation was diluted 100-fold with water before the measurement.

visual evaluation – as soon liposomes are formed after sonication they become transparent due to size reduction usually particles smaller than 100 nm they become transparent and distinguishable transparency are noted.

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8.2.3.2.Encapsulation efficiency

Physical separation performed by sac dialysis in 500 ml chamber filled with water, temperature set to 4C°. samples taken at 1,1.5 and 2 hours, evaluation of liposome before encapsulation and after dialysis can be calculated by the equation:

𝐸𝐸% =𝐿𝑖𝑑𝑜𝑐𝑎𝑖𝑛𝑒 𝑎𝑓𝑡𝑒𝑟 2 ℎ𝑜𝑢𝑟𝑠 𝑒𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑖𝑜𝑛

𝐿𝑖𝑑𝑜𝑐𝑎𝑖𝑛𝑒 𝑏𝑒𝑓𝑜𝑟𝑒 𝑒𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑖𝑜𝑛 ×100

Picture 3. Schematic presentation of modified Franz cell diffusions cell

8.2.3.3.In vitro release test

In vitro release studies were performed using modified Franz diffusion cells with a diffusion area of 1.53 cm2, the test was conducted for 3 or 6 h depending on the experiment, using dialysis cellulose membrane (MWCO 10,000, Medical membranes Ltd). The receptor medium maintained at 32 C◦, stirred at 200 rpm. formulations were applied in the donor compartment and, the donor solution of infinite dosing applied in the donor compartment at pre-determined time points for 3 hours (0.5,1.5,2,3) and for 6 hours (0.5,1.5,2,3,4,5,6 hours) of the receptor medium was collected and replaced with fresh medium. Samples were stored at 25 C◦ until analysis. drug determination was performed by HPLC as previously described.

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Picture 4. Schematic presentation of modified Franz cell diffusions cell

8.3.Statistical optimization

Design expert 7, the statistical software package from the Stat-Ease Inc. is used for design of the experiment, characterization and optimization as well, this tool supplied the design expert matrices for screening all factors required, statistical significance of these factors is established with analysis of variance (ANOVA).

8.4.Statistical analysis

Statistical analysis was performed using IBM SPSS version 20, Microsoft Excel 2013. One-way ANOVA was used for statistical analysis of the various experiments. A p value <0.05 was considered statistically significant. Results were reported as the mean ± standard deviation (SD)

9. RESULTS

9.1.Pre-optimization formulations

Pre-optimization formulations of liposomes preformed initially with different probes tips of sonication device (mentioned above) the liposomes have been sonicated with MS73 and later on switched to TT13, the reason behind switching probes were due to difficulties in sonication and appearance of foam which isn’t appeared after changing to the latter one. The foam made more difficult the analysis of liposomes characterization. Picture 5. Shows the on the left-hand side liposomes before went through sonication and on the right-hand side liposomes after ultrasonic treatment, transparency factor shows that there is reduction in size.

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Picture 5. Un-sonicated and sonicated liposomes

9.1.1. Sonication power

The sonication amplitude was varied in range of 26%-55%, the initial amplitude of pre-formulation was 55% and only later on reduced to the range of 26% - 31%, reason of reduction in intensity is again the appearance of foam and difficulties in characterization, the 30% were found to be the maximal amplitude which liposomes formed without foam and well

characterized with the intention to reduce in size. 9.1.2. Lipoid amount

The amount of lipoid varied in the range of 10 mg/mL to 400 mg/mL examined and formulated, the formulations with different lipoid amounts have been analyzed by size, PDI, pH and ζ-potential, with different sonication probes and finalization conclusion achieved that 100-400 mg/mL selected for further optimization.

9.1.3. Optimization plan

To obtain optimal formulation the amount of the lipoid has been optimized based on the characteristics which based on the design expert factors include entrapment efficiency, in-vitro release test, pH, ζ-potential.

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The goal of EE is to maximize it in order to achieve a larger quantity, entrapment refers to material enshrinement into a carrier and while minimization of release is required and to have a size range of 43-100 nm with minimal PDI so an acceptable result of distribution will be obtained together with ζ-potential so more stable liposomes will be observed.

pH value 6 is more desirable since skin has acidic tendency.

Table 2. characterization pre-optimized formulation

Lipoid (mg) EE (%) Release 1h (μg/mL) Release 3h (μg/mL)

Size (nm) PDI ζ-potential pH

1000 12,4552 5925,75 9641,65 66,21 0,140 -14.2 6,15 1000 29,3315 4986,79 8898,6 58,41 0,382 -16.0 5,78 1501 17,2604 4756,36 7447,28 43,22 0,236 -15.4 5,72 2000.5 8,95425 5483,94 8691,99 44,83 0,245 -15.3 6,1 2500 21,6082 6531,95 2000,5 9430.38 56,44 0,380 -16.1 5,88 2500 8,36574 3221,07 6357,38 69,98 0,376 -17.1 6,00 2999.5 9,57929 11658,3 11369,1 76,27 0,229 -19.5 5,95 3499 23,0723 793,943 1307,43 89,91 0,136 -20.0 5,98 4000 16,8151 2073,28 2809,37 95,23 0,371 -16.8 5,85 4000 7,3945 5462,02 8088,84 114,3 0,100 -10.0 5,97

Optimization factors shown in Table 2 contains the ten formulations with 8 parameters which calculated in order to select the optimized formulation that varies in the range of 100 mg/mL up to 400 mg/mL of the soybean phospholipid with all the factors that are relevant for the

characterization includes the in-vitro release test after 1 and 3 hours respectively.

Size of the formulation fluctuate between 43-114 nm while PDI is in range of 0,100 to 0,382, at the range of 150 mg/mL and 200 mg/mL same PDI achieved, as well as at the 400 mg/mL different PDI and size results calculated, the argumentation here is some results are highly nor reproducible

due to different amounts of the lipoid and yet certain liposomes showed common sizes and ζ-potential such as the 250 mg/mL.

The exception in PDI at the two formulations composed of 400 mg/mL appears to be misleading since PDI of 0,100 achieved but this measurement highly not reproducible due to thickness of the liposome and difficulties of characterization and low attenuation reported by the dynamic light scattering system.

All formulation shows stable pH values the fluctuate from 5,72-6,15 can imply formulations are suitable for further development onto the dermal skin delivery area.

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Certain tendencies can be seen such as increase in size of liposomes with the increment in lipoid amount (figure 1). the phenomena explained by the more lipoid amount the thicker liposome is, and more difficult to cause heterogeneity by ultrasonic manner which leaves liposomes bigger in size. The decisive factor that taken into consideration for optimized formulation is the EE of all ten pre-optimized formulations are varying from 7,3%-29,33% of encapsulation after two hours, (figure 2) shows the efficiencies next to the amount of phospholipid in formulation, when 7,3% as minimal encapsulation resulted with 400 mg/mL and highest occurred at the 100 mg/mL of amount phospholipid.

In-vitro release test manifestation reveals almost all formulations had increase in the release except few of them due to non-reproducible results. major formulations which showed increment by half-fold almost.

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figure 1. Size of liposomes at different amount of phospholipids

figure 2. Distribution of encapsulation efficiencies with different amount of lipoid

0 20 40 60 80 100 120 140 500 1000 1500 2000 2500 3000 3500 4000 4500 S iz e (nm) Lipoid amount (mg) 0 5 10 15 20 25 30 35 500 1000 1500 2000 2500 3000 3500 4000 4500

E

n

cap

su

la

ti

o

n

ef

fi

ci

en

cy

(%

)

Lipoid amount (mg/mL)

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figure 3. Release of lidocaine from various liposomes formulations with different content of phospholipid

Figure 3 gives visualization of the pre-optimized formulations the time versus the flux average with ±SD calculated.

After taking into considerations and based on the analysis of results, optimized amount of 315,1 mg/mL of lipoid calculated for optimization and further experimental investigation of IVRT and EE.

9.2.In-vitro release of optimized formulation results

A triplicate batch with same amount of lipoid with 315 mg/mL with same concentration of 23.11 mg/mL Lidocaine HCl freshly prepared for the significance comparison of release flux after one hour and three hours.1-hour mean flux were 3005,67 μg/cm2 ± 721,40 while 3 hours flux was 5471,23 μg/cm2 ±1548,81.

Concluding the one-hour release test with one-sample T test p-value is less than 0,05 (p<0,05) between batches number one and two which means statistically significant difference.

0.000 20.000 40.000 60.000 80.000 100.000 120.000 140.000 0 0.5 1 1.5 2 2.5 3 C alcula ted flux , (µg /cm²) x 100

Time, h

Form 1, 2500 mg Form 2, 2500 mg Form 3 , 1000 mg Form 4, 2500.5 mg Form 5, 2999.5 mg Form 6, 4000 mg Form 7, 3499 mg Form 8 , 4000 mg Form 9, 1501 mg Form 10, 1000 mg

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Comparison of batch number one and three shows p-value of more than 0.05 (p>0,05) than the batches are not statistically significant. Last comparison of batch number two and three shows same result as previously as p-value is more than 0,05 (p>0,05) than the batches are not statistically significant difference.

Comparison of batches after 3 hours of release show that batch number 1 and batch number two show p-value of less than 0,05 (p<0,05) statistically significant difference between flux. Batch numbers 1 and 3 shows a p-value of greater than 0.05 which means not statistically significant difference, at last comparison between batch number 2 and 3 shows same results as shown between batch 1 and 3 - not statistically significant (p>0,05).

Furthermore, the release profile of the batches examined against lidocaine release as control and for comparison purposes. Observable details seen that the control release is in a greater than batches (figure 4), explained by the reason that lidocaine permeation through membrane occurs straight forward in contrast to lidocaine encapsulated in liposome which has to pass the

phospholipid bilayer.

figure 4. The release of lidocaine from optimized liposomes formulation

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 F lux a ve ra g e (µg /cm 2 ) Time (hours)

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9.3. Encapsulation efficiency of optimal formulations

Drug incorporation is an important factor for drug delivery systems especially when speaking about expensive materials involved. EE of three batches after 2 hours results are fluctuating between 22,28%-35,33%, median is 30,8% with ± SD 7,38%, the result of median of 30,8% is almost twofold higher than the expected by the optimization plan predicated by expert design seven. This results of 30,8% is far from the 78,6% achieved by encapsulation of liposomes at around same size at the experimental work of Wang et al. (42). This high number is achieved by the help of certain enhancers and due to that a significant twofold result of encapsulation is noted, while this work has not applied any external substance to enhance the penetration.

9.4.Stability test of optimized formulation

Stability test were measured for a period of 14 days in 4 points (1,8,10,14), the parameters selected for stability are PDI, pH and size. The comparison is displayed as average with ± standard deviation and comparison done between liposomes containing lidocaine in PBS in contrast to unloaded liposomes solution without lidocaine.

Figure 5 shows the PDI stability of 0,253 ± 0,054 after 14 days relative stability of liposomal lidocaine in contrast to unloaded liposomes which has a certain reduction in PDI after 14 days (0,185 ±0,05) while lidocaine liposome batches had a 0,05 reduction on average.as seen lidocaine increased in PDI which may reflect the better stability of lidocaine for a longer period but this further analysis has to be investigated.

Figure 6 represents the acidity manner over time and clearly seen from the chart that batches of liposomes containing lidocaine were stable at around pH 6 which might indicate their usefulness for future investigation for dermal skin delivery experiments, while again unloaded liposomes showed certain degree of reduction of acidity may hint that the lipophilic lidocaine is stabilize the liposome.

Figure 7 shows the size of lidocaine liposomes averages of three batches kept at the margins of 74-81 nm over the course of 14 days, where, again, unloaded liposomes fluctuated from 90-110,5 nm which shows a larger fluctuating in size.

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Figure 5.PDI comparison stability of liposomes

Figure 6. Acidity stability of liposomes over time course 5 5.5 6 6.5 7 7.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

pH

Days

Lidocaine HCl liposomes Unloaded liposomes

5 5.5 6 6.5 7 7.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 pH Days

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Figure 7. Size stability of liposomes in time

40 50 60 70 80 90 100 110 120 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 S iz e (nm) Days

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DISCUSSION

An effort done to elucidate method for quantitative analysis of lidocaine with establishment delivery system with it and assessment its quality interpretation along with stability, focusing on biopharmaceutical appraisal of lidocaine release profiles.

Finding shows that lidocaine can be incorporated into liposomes system which are meets the literature requirements for liposomes definitions and size varies from 43-114 nm that size can be suitable for developmental of further skin delivery system investigations.

Encapsulation of 30,8% may suggest lower degree to what literature shows, but research here preformed without any skin or membrane enhancers which has to be taken into consideration as done in other publications to increase the encapsulation of lidocaine.

Parameters that has to be taken into consideration is PDI, PH and size in order to have a better stability of the liposomes as well as the PBS will keep liposomes in the skin pH range so further scientific work can examine it in future while this experiment showed that lidocaine as lipophilic primarily determined by the aromatic group can indicate that lidocaine with liposomes is

stabilized by lidocaine in contrast to empty liposomes which is not maintained by PBS solution only. A longer period of stability examination is required.

Lidocaine incorporation in liposomic system is sustained compared to lidocaine solution which released in higher rates due to semi-permeability of the membrane.

Parameters that has to be treated with caution is avoidance of over saturations of phospholipid which can cause thick viscous formulation which might ravage the characterization of liposomal. as well as the amount of lipoid can indicate for bigger size of liposomes although no correlation found. Particular aspect of encapsulation should indicate that liposomal formulation can be further investigated for topical investigation since lidocaine diffusion is not immediate and can be extended efficiently to three hours.

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CONCLUSIONS

1.Lipid Nano carrier systems containing lidocaine were successfully formulated and this is confirmed by particles size, PDI, pH data obtained from three experimental batches of optimized formulations.

2.The stability of formulated liposomal systems with lidocaine was tested for 14 days and satisfactory stability were confirmed, further stability testing is should be performed in order to confirm long term stability of the optimized formulations.

3.Biopharmecutical evaluation of lidocaine release from liposomal systems demonstrated relatively high flux of lidocaine if compared diffusion of lidocaine from solution through semi-permeable membrane.

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RECOMMENDATIONS

1.Stability tests have tested for 14 days which may by insufficient to examine a long-term stability, due to that longer stability of minimal 28 days required.

2.In-vitro release study are not sufficient to estimate reliably the role of liposomal lidocaine HCl

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