Hypercornification Of The Pilosebaceous Duct

Published Date: 02 Nov 2017

Acne vulgaris (cystic acne or simply acne) is a common human skin disease, characterized by areas of skin with seborrhea, comedones, papules, pustules, nodules and possibly scarring. Acne affects mostly skin with the densest population of sebaceous follicles like the upper part of the chest, and the back. The lesions are caused by changes in pilosebaceous units, skin structures consisting of a hair follicle and its associated sebaceous gland.1

The term acne is derived from Greek word "acme" which means "point or edge". Although generally considered to be a benign, self-limiting condition, acne may cause severe psychological problems or disfiguring scars that can persist for a lifetime. The term "acne" refers to the presence of pustules and papules. The most common form of acne is known as acne vulgaris, meaning "common acne". Many teenagers get this type of acne. Use of the term "acne vulgaris" implies the presence of comedones. The term "acne rosacea" is a synonym for rosacea, however some individuals may have almost no acne comedones associated with their rosacea and therefore prefer the term rosacea. Chloracne is associated with exposure to poly-halogenated compounds.2

Propionibacterium acnes (P. acnes) is the anaerobic bacterium species that is widely concluded to cause acne, though Staphylococcus species has been universally discovered to play some role. In particular Staphylococcus, Streptococcus and Candida albicans can infect tiny oil-secreting sebaceous glands found in large numbers on the face, upper back and chest.1

Acne occurs most commonly during adolescence, and often continues into adulthood. In adolescence, acne is usually caused by an increase in testosterone, which accrues during puberty, regardless of sex. For most people, acne diminishes over time and tends to disappear or at the very least decreases by age 25. There is, however, no way to predict how long it will take to disappear entirely, and some individuals will carry this condition well into their thirties, forties, and beyond.1

Some of the large nodules were previously called "cysts" and the term nodulocystic has been used to describe severe cases of inflammatory acne. The "cysts" or boils that accompany cystic acne, can appear on the buttocks, groin, and armpit area, and anywhere else where sweat collects in hair follicles and perspiration ducts. Cystic acne affects deeper skin tissue than does common acne.1

Aside from scarring, its main effects are psychological, such as reduced self-esteem and in very extreme cases, depression or suicide. Acne usually appears during adolescence, when people already tend to be most socially insecure. Early and aggressive treatment is therefore advocated by some to lessen the overall long-term impact to individuals.

1.1.1 SIGNS AND SYMPTOMS

Typical features of acne include: seborrhea (increased oil-sebum secretion), comedones (blackheads and whiteheads), papules (pinheads), pustules (pimples), nodules (large papules) and, possibly scarring. The appearance of acne varies with skin color.

Scars

Acne scars are the result of inflammation within the dermis brought on by acne. The scar is created by the wound trying to heal itself resulting in too much collagen in one spot. Physical acne scars are often referred to as "Icepick" scars. This is because the scars tend to cause an indentation in the skin's surface. Although quite rare, the medical condition Atrophia Maculosa Varioliformis Cutis also results in "acne-like" depressed scars on the face. This are mainly of 4 types as following:

Ice pick scars: Deep pits, that are the most common and a classic sign of acne scarring.

Box car scars: Angular scars that usually occur on the temple and cheeks, and can be either superficial or deep, these are similar to chickenpox scars.

Rolling scars: Scars that give the skin a wave-like appearance.

Hypertrophic scars: Thickened, or keloid scars.

Pigmentation

Pigmented scars is a slightly misleading term, as it suggests a change in the skin's pigmentation and that they are true scars. Pigmented scars are usually the result of nodular or cystic acne (the painful ‘bumps’ lying under the skin). They often leave behind an inflamed red mark. Often, the pigmentation scars can be avoided simply by avoiding aggravation of the nodule or cyst. Pigmentation scars nearly always fade with time taking between three months to two years to do so, although can last indefinitely if untreated.

1.1.2 PATHOGENESIS OF ACNE VULGARIS

Precise mechanisms of acne are not known but there are 4 major pathogenic factors:

1. Increased sebum production,

2. Hypercornification of pilosebaceous duct,

3. Abnormal bacterial function and

4. Production of inflammation

1. Increased sebum production2

Role of sebaceous glands in the pathogenesis of acne has long been recognized, so much so that the disease is standardly classified as a sebaceous gland disorder. However, such a designation is over-simplification.

Pilosebaceous unit, the ‘seat’ of acne, is cell lined follicle with large sebaceous gland and a fine vellus hair that rarely extends out of follicle. These are most common in the acne prone areas such as cheek, nose and forehead and also on the chest and back.

Increased rate of sebum production is one of the most important factors involved in the development of acne lesions. As a group, patients with acne secrete more sebum than normal individuals and severity of acne is related to the degree of seborrhea which is directly dependant on the size and rate of growth of sebaceous glands, which is under the control of androgens. Increased sebum production characteristic of patients with acne is often due to end-organ hyper response. Differential peripheral conversion of testosterone to dihydrotestosterone may explain why glands in sebaceous areas enlarge at puberty. Plasma testosterone levels are usually elevated in females with nodulocystic acne.

Elevated serum levels of free testosterone were noted in acne patients, which correlated with acne lesions. Free testosterone is the only parameter which may be used diagnostically in males. A positive correlation exists between androgens and acne lesions in males. A positive correlation between androgens and acne lesions in females points to the importance of peripheral hormone action in the skin.

Figure 1: Basic mechanisms involved in the pathogenesis of acne. (Journal of Pakistan Association of Dermatologists)

One possible role of sebum in the pathogenesis of acne is its primary or associative role in comedogenesis. Another indisputable role for sebum is providing the substrate for P. acnes growth, specifically triglyceride acted upon by P. acnes lipase to form diglycerides, monoglycerides and free fatty acids from which glycerol, the utilizable moiety for P. acnes metabolism is formed.

2. Hypercornification of the pilosebaceous duct

Obstruction of the pilosebaceous canal precedes the development of acne lesions. The obstruction is produced by the accumulation of adherent keratinized cells within the canal that form an impaction obstructing the flow of sebum. Cause is unknown but the process may be under the influence of androgens. It may also be due to an abnormality in the sebaceous lipids resulting in a relative hyper proliferation of corneocytes. Comedone formation may be due to a localized deficiency of linoleic acid in pilosebaceous duct. Linoleic acid is incorporated via plasma into sebaceous gland cells, where it is diluted due to large volume of sebum and the ductal corneocytes are effectively bathed in an inadequately low level of linoleic acid.2

As the follicular lumen becomes obstructed by abnormally desquamated follicular cells, sebum gets trapped behind the hyperkeratotic plugs, dilating the follicle. Normal follicular architecture is lost at this point. The end result of this hyperkeratinization is the development of comedones {open comedone = blackhead and closed comedone = whitehead: fig. 2}. Microscopically these lesions are dilated pilosebaceous ducts containing a mixture of cornified follicular epithelium, sebum, bacteria and saprophytic yeasts.

Premenstrual exacerbation of acne, a well-recognized clinical feature, can also be explained to some extent by changes in duct size as fluid retention swells keratin.

Biopsy and culture of early non-inflamed lesions show that 30% of these are without bacteria suggesting that ductal bacteria are not needed for initiation of cornification.

Prime defect in acne vulgaris is an increase in sebum excretion, which in turn leads to bacterial colonization and infection, which in turn causes histopathological (and clinical) lesions of acne – a minor component of which is the ductal changes. Ductal blockage theory is a myth that persists in the face of all evidence that suggests the changes interpreted as blockage are secondary. In particular, there is no evidence that comedones are primary lesions and the black headed ones are innocent bystanders on the pathological primrose path to papules and pustules.

Image1

Figure 2: Whitehead and blackhead

3. Abnormal bacterial function

Follicular micro-organisms have a some role in acne. A role of microorganisms in acne has been championed since the beginning of the 20th century. Skin surface in acne prone areas are colonized with Staphylococcus epidermidis and Propionibacterium acnes. Selective inhibitory studies suggest that the main organism is P. acnes.2

The anaerobic P. acnes proliferates in the ideal environment of the comedo: an obstructed lipid rich lumen with decreased oxygen tension. This overgrowth of P. acnes hydrolyses sebum triglycerides, producing free fatty acids which may lead to microcomedo formation.

At present it is not possible to decide whether the microflora are initiators of the lesions or whether they take advantage of the habitat in the lesion, or whether a critical balance of groups of micro-organisms occupying a follicle is the essential factor for the acnegenesis.

thumb_micrcomedo

Figure 3: Microcomedone (http://www.abateit.com/whatisacne/)

4. Production of inflammation

The association of P. acnes proliferation with inflammatory lesions of acne is best borne out with the significant suppression of P. acnes with antibiotic therapy. There is a parallel improvement and decrease in the number of acne lesions. P. acnes contributes to inflammation through activation of various chemotactic factors, as well as, through promoting rupture of the comedo.

It is not clear why the endogenous bacteria colonize the sebaceous follicles. P. acnes elaborate a low molecular weight polypeptide that presumably diffuses through abnormally keratinized follicular epithelium of sebaceous follicles, still physically intact and attract polymorphonuclear leucocytes to follicular site. The intrafollicular P. acnes is ingested by neutrophil, with the consequent release of hydrolytic enzymes that are thought to effect follicular wall dyshesion and rupture. This disruption allows the intrafollicular contents to escape into surrounding dermis and produce inflammation. Experimental evidence suggests that inflammation results from a variety of insults, such as neutrophilic hydrolytic enzymes, P. acnes enzymes, sebum and foreign bodies (Figure 4). The combination of keratin, sebum and microorganism particularly P. acnes leads to release of proinflammatory mediators and accumulation of T-helper lymphocytes, neutrophils and foreign body giant cells. This in turn causes the formation of inflammatory papules, pustules and nodulocystic lesions.

Figure 4: Sequence of events leading to acne inflammation primarily induced by P. acnes (Pochi PE, The pathogenesis and treatment of acne. Ann Rev Med 1990; 41: 187-98.)

Early cellular infiltrate is lymphocytic seen around blood vessels and duct. Within 12-24 hours polymorphonuclear leucocytes appear but lymphocytes remain in the papule as predominant cell infiltrate. Ductal rupture is not a prerequisite for development of inflammation.

The impact of emotional well-being and function can be critical and is associated with depression and higher than average unemployment rates. Proper understanding of pathogenesis of acne will lead to successful acne management which necessitates the ability of treating physician to apply therapy to evolutionary stage of disease. More than one pathogenic mechanism should be targeted for treating acne lesions.

1.1.3 CAUSES

Acne develops as a result of blockages in the follicles. Hyperkeratinization and formation of a plug of keratin and sebum (a microcomedo) is the earliest change. Enlargement of sebaceous glands and an increase in sebum production occur with increased androgen production at adrenarche.

Hormonal

Hormonal activity, such as menstrual cycles and puberty, may contribute to the formation of acne. During puberty, an increase in sex hormones called androgens cause the follicular glands to grow larger and make more sebum. Use of anabolic steroids may have a similar effect. Several hormones have been linked to acne: the androgens testosterone, dihydrotestosterone (DHT) and dehydroepiandrosterone sulfate (DHEAS), as well as insulin-like growth factor 1 (IGF-I).

Development of acne vulgaris in later years is uncommon, although the incidence of rosacea, which may have a similar presentation, is increased in older age groups. True acne vulgaris in adult women may be a feature of an underlying condition such as pregnancy, or disorders such as polycystic ovary syndrome and Cushing's syndrome. Menopause-associated acne (known as acne climacterica) occurs as production of the natural anti-acne ovarian hormones estradiol and progesterone fail, permitting the acnegenic hormone testosterone to exert its effects unopposed.

Genetic

The predisposition for specific individuals to acne is likely explained by a genetic component, which has been supported by twin studies as well as studies that have looked at rates of acne among first degree relatives. The genetics of acne susceptibility is likely polygenic, as the disease does not follow classic Mendelian inheritance pattern. There are multiple candidates for genes which are possibly related to acne, including polymorphisms in TNF-alpha, IL-1 alpha, CYP1A1 among others.

Psychological

While the connection between acne and stress has been debated, scientific research indicates that "increased acne severity" is "significantly associated with increased stress levels." The National Institutes of Health (USA) list stress as a factor that "can cause an acne flare." A study of adolescents in Singapore "observed a statistically significant positive correlation between stress levels and severity of acne." It is also not clear whether acne causes stress and thus perpetuates itself to some extent.

Infectious

Propionibacterium acnes (P. acnes) is the anaerobic bacterium species that is widely concluded to cause acne, though Staphylococcus epidermidis has been universally discovered to play some role since normal pores appear colonized only by P. acnes. Regardless, there are specific clonal sub-strains of P. acnes associated with normal skin health and others with long-term acne problems. It is as yet inconclusive whether any of these undesirable strains evolve on-site in the adverse conditions or are all pathogenically acquired, or possibly either depending on the individual patient. These strains either have the capability of changing, perpetuating, or adapting to, the abnormal cycle of inflammation, oil production, and inadequate sloughing activities of acne pores. At least one particularly virulent strain, though, has been circulating around Europe for at least 87 years.

Diet

A high glycemic load diet is associated with worsening acne. There is also a positive association between the consumption of milk and a greater rate and severity of acne. Other associations such as chocolate and salt are not supported by the evidence. However, chocolate contains a varying amount of sugar that can lead to a high glycemic load, and it can be made with or without milk in one form or another.

1.1.4 DIAGNOSIS

There are multiple grading scales for grading the severity of acne vulgaris, three of these being:

Leeds acne grading technique: Counts and categorises lesions into inflammatory and non-inflammatory (ranges from 0–10.0).

Cook's acne grading scale: Uses photographs to grade severity from 0 to 8 (0 being the least severe and 8 being the most severe).

Pillsbury scale: Simply classifies the severity of the acne from 1 (least severe) to 4 (most severe).

1.1.5 MANAGEMENT

Many different treatments exist for acne including benzoyl peroxide, antibiotics, retinoids, antiseborrheic medications, anti-androgen medications, hormonal treatments, salicylic acid, alpha hydroxy acid, azelaic acid, nicotinamide, and keratolytic soaps. They are believed to work in at least 4 different ways, including: normalising shedding into the pore to prevent blockage, killing Propionibacterium acnes, anti-inflammatory effects, hormonal manipulation.

Medications

Benzoyl peroxide

Benzoyl peroxide is a first-line treatment for mild and moderate acne vulgaris due to its effectiveness and mild side-effects (primarily an irritant dermatitis). It works against the "P. acnes" bacterium, and normally causes just dryness of the skin, slight redness, and occasional peeling when side effects occur. This topical does increase sensitivity to the sun as indicated on the package, so sunscreen should be used during the treatment to prevent sunburn. Benzoyl peroxide has been found to be nearly as effective as antibiotics with all concentrations 2.5%, 5.0%, and 10% equally effective.[31] Unlike antibiotics, benzoyl peroxide does not appear to generate bacterial resistance.

Antiseptics

Sometimes benzoyl peroxide topical medication is combined with a salt of hydroxyquinoline, such as potassium hydroxyquinoline sulphate, which has antibacterial properties. One such topical product is available without prescription in the UK.

Antibiotics

Antibiotics are reserved for more severe cases. With increasing resistance of P. acnes worldwide, they are becoming less effective. Commonly used antibiotics, either applied topically or taken orally, include erythromycin, clindamycin, and tetracyclines such as minocycline.

Hormones

In females, acne can be improved with hormonal treatments. The common combined estrogen/progestogen methods of hormonal contraception have some effect, but the antiandrogen cyproterone in combination with an oestrogen (Diane 35) is particularly effective at reducing androgenic hormone levels. Diane-35 is not available in the USA, but a newer oral contraceptive containing the progestin drospirenone is now available with fewer side effects than Diane 35 / Dianette. Both can be used where blood tests show abnormally high levels of androgens, but are effective even when this is not the case. Along with this, treatment with low-dose spironolactone can have anti-androgenetic properties, especially in patients with polycystic ovarian syndrome.

Topical retinoids

Topical retinoids are medications that normalize the follicle cell life cycle. This class includes tretinoin (Retin-A), adapalene (Differin), and tazarotene (Tazorac). Like isotretinoin, they are related to vitamin A, but they are administered topically and they generally have much milder side effects. They can, however, cause significant irritation of the skin. The retinoids appear to influence the cell life cycle in the follicle lining. This helps prevent the hyperkeratinization of these cells that can create a blockage. Retinol, a form of vitamin A, has similar, but milder, effects and is used in many over-the-counter moisturizers and other topical products. Effective topical retinoids have been in use for over 30 years, but are available only on prescription, so are not as widely used as the other topical treatments. Topical retinoids often cause an initial flare-up of acne and facial flushing.

Oral retinoids

A daily oral intake of vitamin A derivative isotretinoin (marketed as Roaccutane, Accutane, Amnesteem, Sotret, Claravis, Clarus) over a period of 4–6 months can cause long-term resolution or reduction of acne. It is believed that isotretinoin works primarily by reducing the secretion of oils from the glands, but some studies suggest that it affects other acne-related factors as well. Isotretinoin has been shown to be very effective in treating severe acne and can either improve or clear well over 80% of patients. The drug has a much longer effect than antibacterial treatments and will often cure acne for good. The treatment requires close medical supervision by a dermatologist because the drug has many known side effects (many of which can be severe). About 25% of patients relapse after one treatment. The most common side effects are dry skin and occasional nosebleeds (secondary to dry nasal mucosa). There are reports that the drug has damaged the liver of patients. For this reason, it is recommended that patients have blood samples taken and examined before and during treatment. In some cases, treatment is terminated or reduced due to elevated liver enzymes in the blood, which might be related to liver damage. Blood triglycerides also need to be monitored.

Anti-inflammatories

Nicotinamide, (vitamin B3) used topically in the form of a gel, has comparable efficacy to topical clindamycin used for comparison. The benefit of topical nicotinamide seems to lie in its anti-inflammatory nature. It is also purported to result in increased synthesis of collagen, keratin, involucrin and flaggrin. It may be useful for reducing skin hyperpigmentation (dark acne marks), increasing skin moisture and reducing fine wrinkles.

Topical clindamycin is used to treat acne. Clindamycin is in a class of medications called lincomycin antibiotics. It works by slowing or stopping the growth of bacteria that cause acne and by decreasing swelling. It is only for use on the skin.

Ibuprofen is used in combination with tetracycline for some moderate acne cases.

Mandelic acid has been noted to be an effective topical treatment for mild to moderate acne. It is considered to be a gentler alternative to popular alpha hydroxy acids such as glycolic acid and lactic acid.

Alternative medicine

Egg Oil (INCI: Egg Oil) has often been used with success, since it contains antioxidant xanthophylls like Lutein and Zeaxanthin, Cholesterol and long chain polyunsaturated fatty acids (Omega-3, Omega-6) like Docosahexaenoic acid and Arachidonic acid. It has known anti-bacterial, anti-inflammatory properties in skin infections due to the presence of immunoglobulin.

Tea tree oil (melaleuca oil) has been used with some success, where it is comparable to benzoyl peroxide but without excessive drying, kills P. acnes, and has been shown to be an effective anti-inflammatory in skin infections.

Aloe vera: there are treatments for acne mentioned in Ayurveda using herbs such as Aloe vera. However, scientific evidence for the cosmetic effectiveness of Aloe vera is limited and when present is frequently contradictory.

comedone2comedone

closed comedone3 th_cc08

closed comedo (a whitehead): Accumulation of sebum converts a microcomedo into this

th_co24m th_co35th_co08

open comedo (a blackhead): when follicular orifice is opened and distended. Melanin, packed keratinocytes and oxidized lipids forms dark colour.

cysticth_iii823

Cysts Pustules (pimples)

Figure 5: Different type of acnes4

1.2 TOPICAL FORMULATION

A topical medication is a medication that is applied to body surfaces such as the skin or mucous membranes to treat ailments via a large range of classes including but not limited to creams, foams, gels, lotions and ointments. Topical medications differ from many other types of drugs because mishandling them can lead to certain complications in a patient or administrator of the drug.5

Many topical medications are epicutaneous, meaning that they are applied directly to the skin. Topical medications may also be inhalational, such as asthma medications, or applied to the surface of tissues other than the skin, such as eye drops applied to the conjunctiva, or ear drops placed in the ear, or medications applied to the surface of a tooth. As a route of administration, topical medications are contrasted with enteral (in the digestive tract) and parenteral administration (injected into the circulatory system).5

Topical formulations are applied directly to the skin. Advantages of this include:

Increased dose of medication where it is needed

Reduced side effects and toxicity to other organs

Disadvantages include:

Time consuming

At times, complicated e.g. if several different formulations have been prescribed

May be messy or uncomfortable

Topical formulations are made up in a vehicle, or base, which may be optimized for a particular site of the body or type of skin condition. The product may be designed to be moisturizing or to maximize the penetration of an active ingredient, a medicine, into or through the skin.

The amount of the active ingredient that is absorbed through the skin depends on the following factors:

Skin thickness – this varies with body site, age, skin disorder: thin skin absorbs more.

Skin barrier function – horny cell layer disrupted by dermatitis, ichthyosis and keratolytic agents such as salicylic acid and absorbs more.

Skin hydration – up to 10 times more absorption if occluded e.g. skin folds or under a dressing or greasy ointment.

Molecular size of the chemical: small molecules are more easily absorbed.

Whether the chemical is lipophilic – lipophilic agents are better absorbed through the horny cell layer which is made up of lipids including ceramides, cholesterol and fatty acids.

Chemical concentration – stronger concentrations may penetrate more effectively.

Other ingredients in the formulation may interact to increase or reduce potency or absorption rates.

Topical formulations contain an active ingredient, often a medication or drug or botanical, and a vehicle. The vehicle contains water, oil, alcohol or propylene glycol mixed with preservatives, emulsifiers, absorption promoters and fragrances.6

Herbal medication are considered safer than allopathic medicines as allopathic medicines are associated with side effects such as like contact allergy, local irritation, photosensitivity, itching, pruritus, redness, skin peeling, xerosis of the skin. Patients feel more comfortable using topical therapies because they have milder side effects, are easier to use, are generally less expensive and are more readily available. Topical cosmetic formulations are the most preferred treatments asked by patients and are also often most prescribed by family physicians and dermatologists.7

Classification of topical formulations is done in to different categories as solution, lotion, cream, ointment, gel, paste, spray, powder, solid and transdermal patch. Other terms used by cosmetic and pharmaceutical manufacturers include emulsion, paint, suspension, milk, syrup, collodion, balm and mist. Formulae may have mixed ingredients with more than one type of vehicle.6

Nature of the dermatosis

Wet or oozy skin conditions: creams, lotions, drying pastes

Dry scaly skin conditions: ointments, oils

Inflamed skin: wet compresses, soaks followed by creams or ointments

Cracks and sores: bland applications – avoid alcohol and acidic preparations

Site

Palms and soles: ointment or cream

Skin folds: cream or lotion

Hairy areas: lotion, solution, gel, foam

Mucosal surfaces: non-irritating formulations

A successful topical dermatological formulation can be considered to be one that satisfies the target product profile if

Physically and chemically stable (adequate shelf life),

Releases API from the formulation and delivers it into the skin as required for the target indication,

Is cosmetically elegant and acceptable to patients,

Contains only excipients that are necessary, FDA-approved or acceptable from a regulatory perspective, and acceptable for the disease state,

Is easy to apply and compatible with the desired packaging, and

Can be manufactured with a process that is scalable to commercial levels.5

The current research topic, nanogel, is a formulation that satisfies all the above criteria, hence can be considered for the topical delivery of drug.

NANO GEL

Nanogel usually refers to a nanoparticle composed of a cross-linked hydrophilic polymer network (hydrogel). Nanogels are most often composed of synthetic polymers or biopolymers which are chemically or physically cross-linked. Like hydrogels, the pores in nanogels can be filled with small molecules or macromolecules, and their properties, such as swelling, degradation, and chemical functionality, can be controlled.8

Nanogels are usually in the tens to hundreds of nanometers in diameter, by varying solvent quality and branching the volume fraction can be altered variably to maintain a three dimensional structure.8

Traditionally in the name of gels we have heard of semisolid formulations with three dimensional network of organic systems encompassing fluids and drugs. Majorly these systems have been the part of traditional system of topical drug delivery for local effects. Prospects of targeted drug delivery perhaps could not been established with these preparations.9

Various synthetic strategies for the preparation of nanogels include photolithographic and micromolding methods, microfluidics, modification of biopolymers with various approaches, and free radical heterogeneous polymerization in dispersion, precipitation, inverse (mini)emulsion, and inverse microemulsion. In addition, recent advance on controlled/living radical polymerization (CRP) in heterogeneous media to prepare well-defined nanogels with unique properties for drug delivery.10

Release mechanism from nanogel matrices11

The release phenomena, drug release mechanisms from nanogels can be categorized as: i) diffusion-controlled, ii) swelling-controlled, and iii) chemically-controlled. According to Fick's first law of diffusion (with constant or variable diffusion coefficients), the diffusion controlled behavior is the most dominantly applicable mechanism to describe the drug release from hydrogels. The drug diffusion out of a hydrogel matrix is primarily dependent on the mesh sizes within the matrix of the gel, which, in turn, is affected by several parameters, including, mainly, the degree of crosslinking, chemical structure of the composing monomers, and when applicable, type as well as intensity of the external stimuli. Meanwhile, mechanical strength, degradability, diffusivity, and other physical properties of a nanogel network are greatly dependent on its mesh size.

Figure 6: Novel cross-linking methods use in nanogel

Different method of synthesis of nanogels:

1. Emulsification/solvent evaporation method12

Novel nanogels are prepared using either shell cross-linking of polymers, e.g. Pluronic F127 micelles with polyethylenimine (PEI) (F127/PEI nanogel), or penetrating network of polymers, e.g. poly(butylcyanoacrylate) (PBCA) in Pluronic F127 micelles (F127/PBCA nanogel). The hydrophobic core of micelles provides a suitable microenvironment for the incorporation of water-insoluble drugs while the hydrophilic shell formed by EO chains maintains the dispersion stability of Pluronic micelles.

In this method, one polymer is dissolved in organic solvent like chloroform and added dropwise to an aqueous solution of crosslinking agent under stirring. The organic solvent in the emulsion is removed by rotary vacuum evaporation.

Water-in-oil (W/O) heterogeneous emulsion methods10

W/O emulsion methods involve generally two steps: emulsification of aqueous droplets of watersoluble biopolymers in continuous oil phase with an aid of oil-soluble surfactants and crosslinking of biopolymers with water-soluble crosslinkers. The general approaches include inverse (mini)emulsion (often called emulsion crosslinking technique), reverse micelle, and membrane emulsification.

2. Inverse (mini)emulsion method

A W/O emulsion is formed from a mixture consisting of aqueous biopolymer droplets and a continuous oil phase using either a homogenizer or a high-speed mechanical stirrer. Mineral oil and hexane as hydrophobic organic solvents and Span 80 (sorbitan monooleate) and Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) as oil-soluble surfactants were often used to implement colloidal stability of the resulting inverse emulsion. Various drugs, DNA, and cells are physically incorporated into aqueous droplets. The resulting aqueous droplets of biopolymers are then crosslinked with appropriate crosslinking agents. The resulting cross-linked nanogel particles are prepared as dispersion in organic solvents, and thus purified by precipitation, centrifugation, washing with organic solvents such as isopropanol, and lyophilization. By this method, the size of the prepared nanogel particles can be controlled by amount of surfactants and crosslinking agents as well as stirring speed during the formation of inverse emulsion.

3. Reverse micellar method

Similar to the inverse (mini)emulsion method, the reverse micellar method also involves a W/O dispersion; however, a relatively large amount of oil-soluble surfactants is used to form a thermodynamically stable micellar solution consisting of aqueous droplets dispersed in the continuous oil phase. The resulting micellar droplets have a submicron size ranged from tens to hundreds of nanometers in diameter.

4. Membrane emulsification

Membrane emulsification is a relatively new method for the preparation of spherical particles with a highly uniform size distribution. The method involves the use of a membrane, initially Shirasu porous glass (SPG) membrane with a highly uniform pore size ranging from 0.1 to 18 mm, through which a liquid is allowed to permeate under adequate pressure. The resulting uniform-sized droplets are dispersed in the continuous phase, resulting in the formation of simple W/O and O/W emulsions, multiple emulsions such as O/W/O, O/W/O, and solid/O/W dispersions, and the corresponding particles with various shapes including nanospheres, hollow spheres, core–shell nanocapsules, and organic–inorganic hybrid materials.

Figure 7: Illustration of the reverse micellar method for the preparation of nanogels

5. Spray drying method

Spray drying is widely used in pharmaceutical and materials science to prepare capsules, granules, fine powders, and agglomerates. This method involves the use of a spray dryer, mainly consisting of atomizer and drying chamber. Solutions and suspensions of drugs, polymers, and particles are atomized to fine droplets. A stream of hot air induces quick evaporation of solvent from the droplets in drying chamber, resulting in the formation of nanospheres or nanogels. The obtained particles settle into a bottom collector, which are further dried in a vacuum chamber or modified in separated experiments. The size of the resulting microspheres is determined by nozzle size, spray flow rate, atomization speed, and extent of crosslinking.

Chemical crosslinking of dextrans10

Biodegradable dextran (Dex) based nanogels and hydrogels were prepared by various methods based on chemical crosslinking, including carbodiimide coupling, Michael addition, and free radical polymerization. Physical crosslinking based on stereo complexation and self-assembly was also reported for the preparation of nanogels.

6. Dispersion polymerization

Dispersion polymerization is a technique that allows for the preparation of micron-sized particles with narrow size distribution. In the process, most ingredients including monomers, polymeric stabilizers, and initiators are soluble in an organic solvent as a continuous phase. At the onset, polymerization occurs in a homogeneous reaction mixture; however, the formed polymers become insoluble in the continuous medium, ultimately leading to the formation of stable dispersion of polymeric particles with an aid of colloidal stabilizers. The method has been mainly applied to prepare uniform microspheres of hydrophobic polymers including polystyrene (PS) and poly(methyl methacrylate)(PMMA).

7. Precipitation polymerization

Similar to dispersion polymerization, precipitation polymerization involves the formation of homogeneous mixture at its initial stage and the occurrence of initiation and polymerization in the homogeneous solution. As the formed polymers are not swellable but soluble in the medium, the use of cross-linker is necessary to crosslink polymer chains for the isolation of particles. As a consequence, the resulting cross-linked particles often have an irregular shape with high polydispersity (PDI).

8. Inverse (mini)emulsion polymerization

Inverse (mini)emulsion polymerization is a W/O polymerization process that contains aqueous droplets (including water-soluble monomers) stably dispersed with the aid of oil-soluble surfactants in a continuous organic medium. Stable dispersions are formed by mechanical stirring for inverse emulsion process and by sonification for inverse miniemulsion polymerization. Upon addition of radical initiators, polymerization occurs within the aqueous droplets producing colloidal particles. this method has been explored to prepare cross-linked microgels in the presence of difunctional cross-linkers for effective drug delivery. This is due to a facile confinement of water-soluble drugs in aqueous droplets dispersed in continuous organic solvents.

9. Free radical polymerization

Free radical polymerization of methacrylate functionalized dextran allows for the preparation of dextran based hydrogels, as well as nanogels. Various methacrylated dextrans were synthesized by utilizing the hydroxy groups of dextarn that react with different methacrylate precursors. They include methacrylated dextran derivatives of glycidyl methacrylate (Dex-GMA), 2-hydroxyethyl methacrylate (Dex-HEMA), and hydroxy- terminated HEMA-lactate (Dex-LA-HEMA). These monomers are homopolymerized or copolymerized with other monomers.