Microscopy characterisation of micro-and nanosystems for pharmaceutical use

technique for characterising microparticles and nanosystems recently developed by our research group. In particular, polyester or acrylic polymer microparticles for fenretinide administration are presented here. Moreover, with regard to nanosystems, solid lipid nanoparticles, nanostructured lipid carriers for prednisone and clotrimazole administration and monooleine aqueous dispersion are discussed. Methodology Microparticles were produced by the ‘solvent evaporation method’. Solid lipid nanoparticlesand nanostructured lipid carriers were prepared by stirring, followed by ultrasonication. Monooleine aqueous dispersionswere produced by the hydrotrope or by the hot homogenization methods. For scanning electron microscopyanalysis, microparticles were metallized by gold coating (Edwards Sputter coat-ing S150) and visualized at 15 – 20 kV with a 360 Stereoscan (Cambridge Instruments, Cambridge, UK). For Cryo-TEM ­analysis­ samples­were­ vitrified­ and­ transferred to a Zeiss EM922Omega (Zeiss SMT, Oberkochen, Germany). Images were recorded by a CCD digital camera (Ultrascan 1000, Gatan) and analyzed using a GMS 1.8 software (Gatan). Conclusion Microscopy is to be considered as an indispensable tool to study drug delivery systems. In particular, scanning electron microscopy is helpful in giving information about micro-sized powders, allowing to identify microspheres and microcapsules, as well as to obtain size distribution of the observed particles. Introduction Microand nanoparticles have attracted pharmaceutical interest in the last decades since they offer a number of advantages with respect to other delivery systems such as: (a) the ability to maintain unaltered physicochemical characteristics for long periods allowing long-term storage; (b) the possible administration through different ways (oral, intramuscular or subcutaneous) depending on their composition and (c) their suitability for industrial production1. Concerning nanosystems, lipid dispersions possess a potential use as matrixes able to dissolve and deliver active molecules in a controlled fashion, thereby improving their bioavailability and reducing side-effects2. In particular, solid lipid nanoparticles (SLN) are delivery systems in which the nano-dispersed phase has a matrix of crystalline solid lipids. SLN are able to protect encapsulated molecules from degradation and modulate their release3,4. Another generation of SLN is represented by nanostructured lipid carriers (NLC) whose matrix is composed of a mixture of solid–liquid lipids able to better solubilise lipophilic drugs5,6. Another type of lipid dispersion able to provide matrices for sustained­ drug­ release­ is­ typified­ by­ monooleine aqueous dispersions (MAD). MAD are heterogeneous systems generated by the dispersion of an amphiphilic lipid, such as monoolein, in water. They are constituted of complex lyotropic liquid crystalline nanostructures like micellar, lamellar, hexagonal and cubic phases, the predominance of one species over Abstract Introduction This review provides an overview of the use of microscopy as a tool to characterise shape and dimension of micro and nanoparticulate systems. In­ the­ pharmaceutical­ field,­ the­ use of microscopy has been exerted an important role since the advent of micro and nanotechnology. Indeed, the morphology of particles and their­ inner­ structure­ does­ influence­ the modalities of administration and release of encapsulated drugs. Scanning electron microscopy can be employed to study the morphology of dry powders. In particular microparticles made of polymers can be well visualised and their diameters can be measured. By cutting the powder during sample preparation, it is possible to obtain important information about the inner morphology of the microstructures, discriminating either the capsule or the sphere microstructure. Cryo transmission electron microscopy is a precious tool for characterising colloidal systems. In particular, external as well as internal shape of nanoparticulate systems such as solid lipid nanoparticles or lyotropic mesophases can be well identified.­Moreover,­size­of­disperse­ phase and the overall structure of the dispersion can be monitored. We provide an overview about the use of electron microscopy as


Introduction
Micro-and nanoparticles have attracted pharmaceutical interest in the last decades since they offer a number of advantages with respect to other delivery systems such as: (a) the ability to maintain unaltered physicochemical characteristics for long periods allowing long-term storage; (b) the possible administration through different ways (oral, intramuscular or subcutaneous) depending on their composition and (c) their suitability for industrial production 1 .
Concerning nanosystems, lipid dispersions possess a potential use as matrixes able to dissolve and deliver active molecules in a controlled fashion, thereby improving their bioavailability and reducing side-effects 2 .
In particular, solid lipid nanoparticles (SLN) are delivery systems in which the nano-dispersed phase has a matrix of crystalline solid lipids.SLN are able to protect encapsulated molecules from degradation and modulate their release 3,4 .Another generation of SLN is represented by nanostructured lipid carriers (NLC) whose matrix is composed of a mixture of solid-liquid lipids able to better solubilise lipophilic drugs 5,6 .
Another type of lipid dispersion able to provide matrices for sustained drug release is typified by monooleine aqueous dispersions (MAD).
MAD are heterogeneous systems generated by the dispersion of an amphiphilic lipid, such as monoolein,

Introduction
This review provides an overview of the use of microscopy as a tool to characterise shape and dimension of micro and nanoparticulate systems.
In the pharmaceutical field, the use of microscopy has been exerted an important role since the advent of micro and nanotechnology.Indeed, the morphology of particles and their inner structure does influence the modalities of administration and release of encapsulated drugs.
Scanning electron microscopy can be employed to study the morphology of dry powders.In particular microparticles made of polymers can be well visualised and their diameters can be measured.By cutting the powder during sample preparation, it is possible to obtain important information about the inner morphology of the microstructures, discriminating either the capsule or the sphere microstructure.
Cryo transmission electron microscopy is a precious tool for characterising colloidal systems.In particular, external as well as internal shape of nanoparticulate systems such as solid lipid nanoparticles or lyotropic mesophases can be well identified.Moreover,sizeofdisperse phase and the overall structure of the dispersion can be monitored.
We provide an overview about the use of electron microscopy as Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY) the other mainly depending on temperature and water content of the system 7,9 .
Electron microscopy is a precious mean to investigate micro and nanosized pharmaceutical forms.It gives information about shape, inner structure and dimensions of powders and dispersions that couldn't otherwise beidentified.
Scanning electron microscopy (SEM) can be employed to analyse shape, external surface and dimensional distribution of dry microparticles 10 .It can also be used to visualise the inner structure of sectioned microparticles, giving information about the mutual distribution of components, such as polymers and drugs, within the particle.Thanks to thistechnique,itispossibletodefine the analysed micro-object as 'microsphere' or as 'microcapsule' 11 .
For instance, the use of ideal perfectly round polymeric microspheres with exactly the same size circumvents all the disadvantages encountered using powders and granulates.Microspheres are free-flowing and roll without friction, thus no abrasion is present, guaranteeing a dust-free environment.Drugs embedded in the microsphere matrix are released continuously and at a constant rate 12 .
Cryogenic transmission electron microscopy (Cryo-TEM) can be used to analyse liquid dispersions very close to their native state, due to the possibility of vitrifying samples 13 .Nanosized disperse phases that appear as milky liquids by eye, can beaccuratelyidentifiedbycryo-TEM.Uni-or oligo-lamellar vesicles, lamellar phases, cubic phases, cubosomes, exosomes, etc. can be specified and measured one by one 14 .
This article provides an overview about the use of electron microscopy technique as means for characterising microparticles and nanosystems, as recently demonstrated by our research group.In particular with respect to microsystems, polyester-and acrylic polymer-based microparticles are presented here.On the other hand, concerning nanosystems, nanoparticulate dispersions such as SLN, NLC and MAD are illustrated.

Methodology
The authors have referenced some of their own studies in this methodology.The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.

Production of microparticles
Microparticles were produced by the 'solvent evaporation method' 15 .Briefly, 250 mg of a polymer and cationic agent mixture were dissolved in 7.5 ml of CH 2 Cl 2 or ethyl acetate.The mixture was emulsified with 200 ml of an aqueous phase containing 2% (w:v) of 88% hydrolysed polyvinyl alcohol (PVA) (Airvol 205, Air Products, PA, USA) as the dispersing agent.The obtained emulsion was maintained under continuous stirring with a three-blade turbine impeller Eurostar Digital (Ika Labortechnick, Germany) at 700 rpm and by heating (hot plate RCT basic, Ika Labortechnick, Germany) at a temperature higher than the solvent evaporation temperature (40°C in the case of methylene chloride and 77°C for ethyl acetate).At different time intervals, samples were observed microscopically up to complete evaporation of solvent, usually occurring in 3-5 h.Microparticles werethenisolatedbyfiltration.

Production of nanoparticles
SLN and NLC were prepared by stirring followed by ultrasonication 16 .Briefly, 1 g of lipidic mixture was melted at 75°C.The lipidic mixture was constituted of tristearin (in the case of SLN) or tristearin/caprylic/ capric triglycerides (Miglyol 812) 2:1 w/w (for NLC).The fused lipid phase was dispersed in 19 ml of an aqueous poloxamer 188 solution (2.5% w/w).The emulsion was subjected to ultrasonication (Microson TM, Ultrasonic cell Disruptor) at 6.75 kHz for 15 min and then cooled down to room temperature by placing it in a water bath at 22°C.NLC dispersions were stored at room temperature.
In the case of drug containing nanoparticles, the drugs were added to the molten lipidic mixture and dissolved before addition to the aqueous solution.

Production of MAD
MAD were alternatively produced by the hydrotrope or by the hot homogenisation methods.
The hydrotrope method is based on the solubilisation of monooleine in ethanol and its subsequent dilution in a surfactant solution 17 .In particular, 5 ml of an ethanolic solution of monooleine (10% w/w, with respect to total weight of the dispersion), was slowly added to an aqueous solution of Poloxamer 407 (10% w/w) under stirring at 1500 rpm for 90 min, until complete ethanol evaporation.

SEM analysis
For the electronic analysis, microparticles were metallised by gold coating (Edwards Sputter coating S150) and analysed at 15-20 kV by a SEM 360 Stereoscan (Cambridge Instruments, Cambridge, UK) 19 .The vitrified specimen was transferred to a Zeiss EM922Omega (Zeiss SMT, Oberkochen, Germany) TEM using a cryoholder (CT3500, Gatan, Munich, Germany).Sample temperature was kept below 100 K throughout the examination.Specimens were examined with doses of about 1000-2000 e/nm 2 at 200 kV.Images were recorded by a CCD digital camera (Ultrascan 1000, Gatan, Germany) and analysed using GMS 1.8 software (Gatan, Germany).

Discussion
SEM can help in giving information about the influence of technological parameters on the shape of microspheres.
The morphology of microsystems can influence the release behaviour of the encapsulated drug.In particular SEM enables to discriminate the surface aspect of microspheres focusing on the presence or the absence of pores, indeed the porosity is a factor that can promote drug release 21 Moreover, by cutting the sample before the visualisation, it is possible to obtain cross-sectioned microparticles and to observe their inner structure, allowing to discern between a microsphere and a microcapsule 22 .It is well known that a microsphere is a solid matrix type particle whereas a microcapsule has an inner core (reservoir) and an outer shell.The active ingredient in a matrix particle is typically dissolved or melted.Whereas the microcapsule core, either liquid or solid, usually contains the active ingredient 1 .
It is well known that the release of the active constituent is an important parameter depending on the nature of both the polymer used in the preparation as well as the active drug.
Particularly, the release of drug frommicroparticlesisinfluencedby structure or micro-morphology of the carrier and the properties of the polymer itself [23][24][25] .
Drug release from the microparticles can be understood by  considering the geometry of the carrier.The geometry of the carrier (i.e.whether it is reservoir type where the drug is present as core, or matrix type in which drug is dispersed throughout the carrier) governs overallreleaseprofileofthedrugor active ingredients 26 .
Depending on the type of polymer and on the method of production, different type of microparticles can be achieved 27 .
Figure 1 reports SEM images of DL-PLGA 50:50 microparticles obtained by the solvent evaporation method.One can observe the round shape and the smooth surface of the particles; moreover from Figure 1A it is possible to obtain a dimensional distribution by measuring the mean diameters of the particles.
In Figure 2 are reported images showing external and internal morphology of empty DL-PLGA 50:50 microparticles produced by the solvent evaporation method, using as solvent methylene chloride (Figure 2A and B use of methylene chloride allows the production of mononucleate microcapsules with a classic reservoir structure, while the particles by ethyl acetate show a multinucleate structure.This result can be attributed to the different evaporation rates of the solvents; in fact, methylene chloride (boiling point 40°C) evaporates more rapidly than ethyl acetate (boiling point 77°C).
Figure 3 reports external and internal morphology of fenretinide containing DL-PLGA 50:50 microparticles produced by the solvent evaporation method using as solvent ethyl acetate.One can observe that the presence of fenretinide doesn't modify the external morphology and surface, being similar to those reported in Figure 2C.
On the contrary the cross-section, reported in Figure 4B reveals that in the presence of fenretinide the inner morphology of the microparticles is more compact with respect to the microcapsular reservoir structure of the plain microparticles (Figure 2D).
Figure 4 shows external morphology of empty Eudragit RS 100 microparticles produced by the solvent evaporation method using as solvent ethyl acetate.From Figure 4A it is possible to obtain the dimensional distribution of microparticles, ranging from 5 to 40 µm, with a mean diameter of 27 µm.Figure 4B evidences the round shape and the porous surface of microparticles, differently from DL-PLGA ones.
In the case of nanosized liquid systems, cryo-TEM is an optimal method to obtain structural information, revealing morphology and inner details of the samples studied 13 .Figure 5 shows cryo-TEM images of SLN and NLC dispersions constituted of tristearin and tristearin-Miglyol 812 (2:1), respectively.In particular, Figure 5A shows an image of plain SLN, Figure 5B refers to plain NLC, while Figure 5C and D, respectively show prednisone and bromocriptine containing NLC.
The three-dimensional particles are projected in a two-dimensional (2D) way.In Figure 5A-D, depending on the angle of observation in the vitrifiedsamples,theparticlesappearas deformed hexagonal, elongated circular platelet-like crystalline (SLN/NLC top views) or 'needle'-like structures edge-on viewed (SLN/NLC side views).The measured thickness of nanoparticles is between ca. 5 and 40 nm, according to the number of layers in the platelets.However, the exact thicknessisdifficulttomeasuresincethetilt of the particles can't exactly be determined.Figure 5B, C and D, besides the presence of hexagonal and circular NLC top views, the 'needles' are more elliptically shaped, with respect to the SLN side views (Figure 5A).This may be attributed to the presence of Miglyol 812, which is an oil, forming compartments sticking to the surfaces of the nanoparticles solid matrix 28,29 .This behaviour is clearly noticeable in the presence of prednisone and bromocriptine (Figure 5C and D).
Figure 6 shows cryo-TEM images of NLC dispersions constituted of tristearin-Miglyol 812 in the presence (Figure 6A) and in the absence of clotrimazole (Figure 6B).One can observe elongated ovoidal and circular platelet-like crystalline particles in both Figure 6A and B. The nanoparticles edge-on viewed appear as rectangles with stacked tristearin lamellas in Figure 6A, while in Figure 6B the presence of clotrimazole affects nanoparticles aspect, resulting in 'UFO' profile,asshownintheinsets.
Figure 7 shows two aqueous dispersions of monooleine produced in the presence of P oloxamer 407 obtained by the hydrotrope (Figure 7A) or by the hot homogenisation method (Figure 7B).Some authors have reported that the method resulted in the production of cubosome dispersions.Nonetheless in this case only differently shaped vesicular structures mainly unilamellar or with some invaginations were obtained 17,30 .Whereas the dispersion reported in Figure 7B displays the coexistence of vesicles and spherical particles with a homogeneous ordered inner structure typical of cubosome.
It is interesting to note the presence of vesicular structures attached on the surface of cubosomes, as found in many cryo-TEM and X-ray studies, suggesting that through time, a transformation may take place from conglomerates of partially fused vesicles to well-ordered particles 18,31 .
Considering a number of pictures taken from cryo-TEM observation, dimensions can be measured by a scale bar, finding that two different populations are often present: one constituted of large cubosomes (dimensions over 200 nm) and another of smaller cubosomes and vesicles with dimensions about 100 nm and below.
Figure 8 reports two cryo-TEM images of a monooleine/Poloxamer 407 dispersion, evidencing the presence of vesicles and ordered structures.
Fast Fourier transform (FFT) easily enables to obtain an optical diffractogram similar to an electron diffraction pattern.In this way, periodic or repeatable distances in the mesophase structure could be detected, together with the symmetry of the motif.FFT is, therefore, a precious means to identify the different type of liquid crystalline structures 13,32 .For instance, according to the different internal morphologies shown in Figure 8, FFT allowed to evidence two different patterns.In Figure 8A, the first corresponds to a rectangular symmetry with 2D lattice parameters of 7.1 × 10.2 nm (labelled a) while the second corresponds to a 2D hexagonal symmetry with a 7.1 nm lattice parameter (labelled b).
Samples were vitrified as described in a previous study by Esposito et al. 20 Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Esposito E, Drechsler M, Cortesi R. Microscopy characterisation of micro-and nanosystems for pharmaceutical use.OA Drug Design & Delivery 2013 Aug 01;1(1):2.

Figure 2 :
Figure 2: SEM images showing external and internal morphology of plain DL-PLGA 50:50 microparticles produced by the solvent evaporation method, using as solvent methylene chloride (A and B) or ethyl acetate (C and D).
) or ethyl acetate (Figure 2C and D).It is interesting to note that methylene chloride induces the formation of irregular shaped microparticles with different dimensions, whereas the use of ethyl acetate results in round microparticles with smooth surface and homogeneous dimensions.Moreover, analysing the crosssection, it can be noted that the Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Esposito E, Drechsler M, Cortesi R. Microscopy characterisation of micro-and nanosystems for pharmaceutical use.OA Drug Design & Delivery 2013 Aug 01;1(1):2.

Figure 7 :
Figure 7: Cryo-TEM images of monoolein-Poloxamer 407 dispersions obtained by the hydrotrope (A) and by the hot homogenization (B) methods.

Figure 8 :
Figure 8: Cryo-TEM images of cubosomes dispersions.The insets show the FFT lattice parameters of the indicated nanostructures.
Competing interests: none declared.Conflict of interests: none declared.All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Esposito E, Drechsler M, Cortesi R. Microscopy characterisation of micro-and nanosystems for pharmaceutical use.OA Drug Design & Delivery 2013 Aug 01;1(1):2.