extfieldsofstudy: Biology Medicine

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Isoforms, structures, and functions of versatile spectraplakin MACF1

Spectraplakins are crucially important communicators, linking cytoskeletal components to each other and cellular junctions. Microtubule actin crosslinking factor 1 (MACF1), also known as actin crosslinking family 7 (ACF7), is a member of the spectraplakin family. It is expressed in numerous tissues and cells as one extensively studied spectraplakin. MACF1 has several isoforms with unique structures and well-known function to be able to crosslink F-actin and microtubules. MACF1 is one versatile spectraplakin with various functions in cell processes, embryo development, tissue-specific functions, and human diseases. The importance of MACF1 has become more apparent in recent years. Here, we summarize the current knowledge on the presence and function of MACF1 and provide perspectives on future research of MACF1 based on our studies and others. [BMB Reports 2016; 49(1): 37-44]

INTRODUCTION
Cytoskeleton in most multicellular organisms is a highly organized and interconnected network of filaments composing of microfilaments (F-actin), microtubules (MTs), and intermediate filaments (IFs). Cytoskeletal network acts as a cytoplasmic scaffold, defining the cell shape, cell mechanical properties, and functions of many other cellular events, including cell polarization, migration, division, adhesion, intracellular trafficking, and organelle locomotion. Thus, cytoskeleton is both static and adaptive, providing a dynamic cellular architecture that can remodel itself to respond to cellular changes. The tight organization and dynamics of these filaments strongly depends on their associated proteins. It has been demonstrated that spectraplakins are cytoskeletal crosslinkers with ability of interacting with all three types of cytoskeletal filaments, i.e., F-actin, MTs, and IFs (1).
In this paper, basic molecular characteristics of MACF1 are introduced and current knowledge on MACF1 is reviewed by highlighting its versatile functions. In addition, perspectives on future research of MACF1 are provided based on studies by us and others to provide researchers the current status of MACF1 research.

Gene and isoforms of MACF1
MACF1, a large protein with molecular weight of ∼600 kD, was first discovered by Byers et al. in the effort to screen for additional members of the actin crosslinker superfamily (13). They isolated a partial cDNA of MACF1 using degenerate primer-mediated PCR and named it ACF7. Subsequently, murine ACF7 was further characterized (14) and its full-length cDNA encoding a 608-kD protein was cloned (15). Due to the associ- ation of murine ACF7 with both actin and MTs, ACF7 was renamed MACF to represent microtubule actin cross-linking factor. Meanwhile, human cDNA was cloned independently by two groups and named macrophin and trabeculin-, respectively (16,17).
MACF1/ACF7 is encoded by MACF1 gene located on human chromosome 1p32 or mouse chromosome 4 (13,14,18). Human MACF1 gene contains at least 102 exons and spans over 270 kb. Genomic organization analysis of human MACF1 gene has demonstrated that MACF1 and ACF7 are the same gene (18). MACF1 gene is a hybrid of genes encoding plakins and spectrins/dystrophins (15,18). MACF1 shares similar gene sequences with plectin, a well-characterized plakin. It has the same exon-intron boundaries for N-terminal actin-binding domain (ABD), one large exon encoding plectin repeats, and a similar serine/glycine-rich C-terminus containing GSR repeats. In addition, the spectrin repeats in MACF1 are similar to those of dystrophin gene. This hybrid gene structure of MACF1 reveals that it is related to Drosophila gene shot and Caenorhabditis elegans gene vab-10. Due to the hybrid structure of these genes and their combined features of both spectrin and plakin family members, they were assigned to the spectraplakin family (3).
Like dystonin/BPAG1 (19,20), MACF1 displays isoform diversity. Several MACF1 isoforms ( Fig. 1) produced by alternative splicing and different promoter usage have been reported. Three murine ACF7/MACF1 isoforms with a common 3' sequence but a unique 5' region were first identified by Bernier et al. (14). Isoforms ACF7-1 and ACF7-2 contain identical actin-binding domains (ABDs) but different 5'UTRs, while isoform ACF7-3 contains a unique 5'UTR and a longer N-terminal sequence with just the second half of the ABD (14). Subsequently, a fourth isoform MACF1-4 was cloned (18). Different from the three MACF1 isoforms mentioned above, MACF1-4 has no ABD at the N-terminus. Instead, it has plectin repeats ( Fig. 1). Later, another alternatively spliced murine isoform of MACF1 encoding a gigantic protein of approximately 800-kD was revealed (21). It was named MACF1b with the original isoform renamed as MACF1a. MACF1b harbors extra plakin repeats between the plakin domain and the spectrin repeats of MACF1a ( Fig. 1). With different N-terminal domains, the first three isoforms ACF7-1, ACF7-2, and ACF7-3 were renamed as MACF1a1, MACF1a2, and MACF1a3, respectively (21). While studying the role of MACF1 in the nervous system by adopting MACF1a conditional knockout (cKO) mice, a novel isoform MACF1c was found by Goryunov et al. (22). MACF1c is identical to MACF1a except that it has no N-terminal ABD domain (Fig. 1).

MACF1c
Nervous system Plakin, Spectrin repeats, EF hand, and GAR. In addition, a putative MACF/MACF1 isoform MACF2 has been reported by Sun et al. (23). Based on sequence homology analysis, a partial human cDNA clone (KIAA0728) encoding a polypeptide sharing 68% amino acid identities with human MACF was found and called MACF2. However, they found differences in chromosomal locations and nucleotide sequences between MACF and MACF2 transcripts based on unigene database, suggesting that human MACF and MACF2 represent two distinct protein products from two different genes (23). Consequently, MACF2 was demonstrated to be an isoform of BPAG1 and renamed as BPAG1-a (20). Therefore, up to date, a total of six isoforms of MACF1 (MACF1a1, MACF1a2, MACF1a3, MACF1-4, MACF1b, and MACF1c, Fig. 1) have been reported.

MACF1's tissue distribution
As a cytoskeletal linker protein, MACF1 is ubiquitously expressed in different tissues with some isoforms having different distributions (Table 1).
Bernier et al. were the first to show that MACF1 was widely expressed in postnatal mouse tissues, including brain, spinal cord, spleen, liver, heart, skeletal muscle, stomach, lung, kidney, and skin, with the strongest expression in the lung, followed by brain, spinal cord, cardiac/skeletal muscle, and skin (14). They further demonstrated a predominant expression of MACF1 in neural, muscle, and lung tissue at the beginning of embryonic development which was continued into adulthoods (24). A striking difference in tissue distribution for different MACF1 transcripts has been observed. Relatively higher levels of MACF1a2 have been detected in the brain, spinal cord, and lung, while relatively lower levels of MACF1a2 have been found in the kidney, heart, and skeletal muscles without any detection in the skin, liver, stomach, or the spleen. In contrast, MACF1a1 is predominantly found in the skin, kidney, and stomach. Similarly, MACF1a1 mRNA is detected in embryos from day 7.5 to day 10.5, whereas MACF1a2 mRNA becomes detectable only at day 10.5. MACF1a3 is predominantly detected in the brain and spinal cord. Moderate levels of MACF1a3 has been found in the skin, lung, and kidney with-out expression in the heart, skeletal muscle, or the liver (24). MACF1b is expressed in all tissues and throughout the development of mouse embryo (21).
MACF1 is widely expressed in human tissues, including pituitary, adrenal, thyroid, salivary gland, mammary glands, pancreas, heart, and skeletal muscle at different levels (13,16,17). MACF1a2 is highly expressed in the brain, heart, lung placenta, liver, kidney, and pancreas (16), while the hybridization signals of MACF1-4 are visible in all tissues, with the strongest signals in the heart, lung, pituitary gland, and placenta (18).

UNIQUE DOMAIN STRUCTURE OF MACF1
Generally, the structure of MACF1 contains three main domains: an N-terminal domain containing an ABD and a plakin, a rod domain composed of spectrin repeats, and a C-terminal domain consisting of EF-hand calcium-binding domain and a GAS2-related protein (GAR) domain (23). Seven types of functional domains presenting different combination in different isoforms ( Fig. 1) have been found in MACF1.

Actin-binding domain
MACF1 has an ABD located at the N-terminus, a conserved structure among spectraplakins inherited from members of the spectrin superfamily (15,25). ABD consists of two calponin homology (CH) domains CH1 and CH2 that can bind F-actin and enable spectraplakins to interact directly with the actin cytoskeleton. CH1 alone can bind to actin. CH2 exerts a weaker binding affinity for actin. However, CH1-CH2 tandem domain has high binding affinity (26,27). Not all MACF1 isoforms have CH1 and CH2 domains. MACF1a3 only has CH2 domain while MACF1-4 and MACF1c have no CH domain (Fig. 1). Regardless of the CH domain, the interaction of MACF1 with F-actin is directly affected (15,25). Although the crystal structure of MACF1 has not been reported yet, structure analysis of ABD in dystrophin and utrophin spectrins has demonstrated an interdomain linker between the CH1 and CH2 domains, with the interdomain linker primarily determining the structural stability of tandem CH domains (28).

Plakin domain
Following the ABD at the N-terminal of MACF1, there is a plakin domain (23). The plakin domain is also a feature of members of the plakin superfamily (3). It is characterized by a high -helical content (29). Structure analysis of BPAG1 and plectin has revealed a number of spectrin repeats in the plakin domain (30,31), suggesting that the plakin domain is derived from spectrin repeats. The plakin domain is responsible for binding to various adhesion and signaling molecules (e.g. 4 integrin, BPAG2, and Erbin) (32)(33)(34)(35). It plays a role in the mediation of the binding to cell junctions (29). However, the structure and the function of the plakin domain in MACF1 require further investigation.

Spectrin repeats
MACF1 contains both the spectrin repeats and the plakin domain. It is classified as a spectraplakin. Spectrin repeats are the typical architecture of the spectrin family. MACF1 has 23 dystrophin-like spectrin repeats (15). Each repeat contains 110∼120 residues that are folded into three  helices. These helices then form an antiparallel three-helical coiled coil (36,37). These -helical spectrin repeats then form an extended rod-like structure and act as a spacer region that separates different functional domains at the N-and C-termini. In addition, these spectrin repeats endow the protein MACF1 with flexibility (1, 3).

EF hand
MACF1 contains two EF-hand calcium-binding motifs located at the C-terminus that are conserved within the spectrin superfamily (23). Sun et al. have indicated that the EF-hand motifs of MACF1 have no effect on the ability of MACF1 to interact with MTs (23).

GAS2-related protein (GAR) domain
Following the EF-hand motifs, there is a GAR domain located at the C-terminal of MACF1 (Fig. 1). The GAR domain is restricted to spectraplakins. It can be used to distinguish spectraplakins from spectrins and plakins (3). The GAR domain is the homology region shared between Gas2 (growth arrest-specific protein 2) and GAR22 (Gas2 related on chromosome 22) proteins. It has the function to bind and stabilize MTs (15,23).

Plakin repeat domain (PRD)
PRD is unique to members of the plakin family. It only exists in the MACF1b isoform (21). Based on well-characterized A, B, and C type PRDs in desmoplakin (38, 39), Lin et al. have revealed five PRDs in MACF1b, including two A type, one C type, one incomplete B type, and one incomplete A type (21). They have further demonstrated that PRD targets MACF1b to the Golgi complex via its N-terminal portion of two consecutive A-type subdomains.

Plectin repeats
Plectin repeats are unique to the MACF1-4 isoform (18). MACF1-4 contains eight plectin repeats instead of ABD at the N-terminus (Fig. 1). Studies from the plakins that contain plectin repeats have demonstrated the essential role of plectin repeats in binding IFs. Therefore, the same function of plectin repeats may exist in MACF1-4. This requires further study.

MACF1 IN CELL MIGRATION
MACF1 has a well-known function in cell migration by coordinating F-actin and MTs dynamics (5-7). MACF1 binds to both F-actin and MTs with its ABD and MT-binding domains. In endodermal cells, MACF1 can bind along MTs, especially at the plus-ends of those MTs toward the edges of migrating cells (5,25). Loss of MACF1 causes perturbations in MTs trajectories, dynamic stabilities, and tracking ability along actin cables, which can affect cell migration in response to wounding (5). MACF1 deficiency compromises the targeting of MTs along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration (6). The underlying mechanisms involve the F-actin binding domains in MACF1 and an intrinsic actin-regulated ATPase domain of MACF1 for the regulation of cell migration. Therefore, there is a new functional domain in MACF1. MACF1 also sustains directional cell migration in stem cells responsible for homeostasis and wound healing (7). It has been demonstrated that GSK3 can phosphorylate and uncouple MACF1 from MTs during the regulation of cell migration by MACF1 (7). Moreover, MACF1 can regulate cell migration in neurons (8) with several partners, including ErbB2 receptor and ELMO (40,41).

MACF1 IN CELL SIGNALING
Given the implication that MACF1 −/− mice and Wnt3 −/− and Lrp5/6 double-knockout mice have similar phenotype, the role of MACF1 in Wnt/-catenin signal transduction has been determined by Chen et al. (11). Their results have indicated that MACF1 can interact with the Axin complex, including Axin, GSK-3, -catenin, and APC. They have also revealed that MACF1 is responsible for the translocation of the Axin complex from the cytoplasm to the cell membrane where MACF1 also binds to co-receptor LRP5/6. Reduction of MACF1 will impair the translocation of the Axin complex, thus inhibiting Wnt-induced TCF/-catenin-dependent transcriptional activation. In addition, MACF1 has a phosphorylation site for GSK-3 (7). It can also mediate GSK-3 signaling (8). These findings have demonstrated a role of MACF1 in cell signaling. In addition, MACF1 functions in vesicles transport. In axonal vesicle transportation, MACF1 acts as a medium to transport vesicles from the trans-Golgi network to Kif5A. Malfunction of MACF1 can cause vesicles incapable of being mobilized to the cell periphery (42). Interaction between Trans-Golgi protein p230 and MACF1 can mediate the transport of mAtg9 from the trans- Golgi network to peripheral phagophores in the early step of autophagy (43).

MACF1 IN EMBRYO
As a ubiquitously-expressed protein, MACF1 is present at embryonic day 7.5 (E7.5) in mouse (24), which is quite early, suggesting that MACF1 may act as a critical molecule during embryo development. Chen et al. have confirmed the essential role of MACF1 in embryo development using MACF1 −/− mice (11). The MACF1 −/− mice died at the gastrulation stage with a "head-without-trunk". They had developmental retardation at E7.5, lacking primitive streak, node, and mesoderm, similar to the phenotypes of Wnt3 −/− embryos (44) and LRP5/6 double-knockout embryos (45). Moreover, a deletion mutation in the magellan gene encoding MACF1 in zebrafish caused oocyte polarization failure (46). Taken together, these data demonstrate the importance of MACF1 in embryo development.

MACF1 IN THE NERVE
High levels of MACF1 and significant physiological function of the homolog of MACF1 Kakapo/Shot in the nervous system (15,47) indicate that MACF1 might play important role in the nervous system. By generating a nervous system-specific Macf1 knockout (cKO) mouse model, Goryunov et al. (22) have underscored the importance of MACF1 in nervous system development. MACF1 cKO brains have been found to have multiple neuronal developmental defects (22). Defects in neuronal migration in the cKO cortex are also observed, suggesting a critical role of MACF1 in neuronal migration (22). In addition, the MACF1c isoform that lacks an ABD compared to MACF1a cannot compensate for the loss of MACF1a (22). Their findings indicate the necessity to interact with actin for MACF1's function in the nervous system. Other studies have confirmed the role of MACF1 in the regulation of neuronal growth, survival, and migration (8,48,49). These evidences indicate the importance of MACF1 in the nerve.

MACF1 IN SKIN
The physiological function of ACF7/MACF1 in mammalian skin has been uncovered mainly by Elaine Fuchs' group. Karakesisoglou et al. first showed the expression of MACF1 in the epidermis of mice and identified the functional binding sites of MACF1 for both actin and MTs (25). Subsequently, Wu et al. elucidated the function of MACF1 in the skin by conditionally knocking out (cKO) MACF1 in epidermal cells (6,7). No gross morphological change in the skin or hair coat was observed in MACF1 cKO mice. However, a significant delay in repairing full-thickness wounds due to defects in epidermal cell migration was detected in the skin of cKO mice in response to injury. Further studies revealed that cell migration defects were rooted in perturbations in epidermal-ECM adhe-sion caused by a malfunction in focal adhesion dynamics due to MACF1 absence (6). Moreover, a link between MACF1 and GSK3 controlling skin stem cell migration and polarized locomotion upon injury was identified (7). Taken together, these results suggest a key role of MACF1 in maintaining skin integrity and regulating epidermal cell migration through coordinating with F-actin and MTs dynamics. Recently, MACF1 has been linked to cellular processes involved in human epidermal cells in response to extremely-low-frequency electric fields (50).

MACF1 IN BONE
MACF1 is a widely expressed protein. However, its role in bone tissue is not well understood. Our group have focused on the role of MACF1 in bone and reported some primary findings in osteoblasts. MACF1 is widely expressed in osteoblastic cells. It partially co-localizes with F-actin and MTs. It is scattered in the cytoplasm and cell periphery. MACF1 also participates in the osteoblast in response to environmental stimuli (51). Under diamagnetic levitation conditions, the distribution of MACF1 in osteoblasts was altered and the co-localization of MACF1 with F-actin and its co-alignment with MTs were also changed (51). Our further studies have demonstrated that MACF1 is crucial for maintaining cell shape and cell proliferation in osteoblasts (52). Knockdown of MACF1 can induce large cells with a binuclear or multinuclear structure, disrupt the organization of F-actin and MTs, and inhibit osteoblast proliferation (52). These data indicate that MACF1 is important in osteoblasts and bone.

MACF1 IN OTHER TISSUES
MACF1 also plays important roles in the heart, colony, and other tissues (53,54). Fassett et al. (53) have identified a role of MACF1 in regulating cardiomyocyte MTs distribution and adaptation to hemodynamic overload for the first time using inducible cardiac-specific MACF1 knockout (KO) mice. The heart size or function of MACF1 KO mice was not affected under basal conditions. However, exacerbated pressure overload induced left ventricular (LV) hypertrophy, LV dilation, and contractile dysfunction (53). This manifestation has been found to be related to altered MT distribution and several relevant signaling proteins caused by the absence of MACF1 (53). MACF1 is also critical to the maintenance of colonic paracellular permeability because cKO of MACF1 causes significant decrease in colonic mucosal permeability associated with disturbed epithelial arrangement and cytoskeleton dysregulation (54).

MACF1 AND HUMAN DISEASE
In 2014, a human disease caused by a mutation in MACF1 was reported (12). This disease was found in a 12-year-old boy (the proband) who suffered from a skeletal muscle problems with mainly manifestation of generalized hypotonia and pain. Genetic analysis revealed a single duplicated region on chromosome 1p34.3 covering a large part of the MACF1 gene. This affected all 4 major MACF1 isoforms in the proband. Further studies demonstrated that the duplication resulted in a remarkable reduction in MACF1 expression, resulting in reduced motility and internal structural changes of endothelial and satellite cells in the skeletal muscle in the proband. This novel myopathy caused by the duplication in MACF1 was named spectraplakinopathy type 1 (12). The discovery of this MACF1-related genetic human disease has shed light on the importance of MACF1 in human.
Recently, it was found that MACF1 was involved in cancer. A genetic alteration of MACF1 has been detected in numerous cancers, including breast cancer, colorectal cancer, gliomas, and others cancers (55)(56)(57)(58)(59). Although the function of MACF1 in cancer requires further investigation, these data suggest that MACF1 might be used as a marker for cancer diagnosis and cancer therapy.

CONCLUSIONS AND FUTURE PERSPECTIVES
In this review, we summarized the current knowledge of MACF1. As an extensively studied mammalian spectraplakin, MACF1 is broadly expressed with versatility. It has different isoforms, structures, and functions. Similar to other spectraplakins, MACF1 has several isoforms expressed in different tissues. MACF1 participates in many cellular processes and specific-tissue functions. MACF1 is crucial to cell polarization and migration by regulating the dynamics of F-actin and MTs. In addition, MACF1 plays a key role in mediating cellular signaling transduction. The embryonic lethality due to a lack of MACF1 has demonstrated its essential role in embryo development. In addition, MACF1's ubiquitous expression in all tissue indicates its physiological significance. In fact, MACF1 plays critical roles in controlling the normal development of the nervous system, maintaining skin integrity and colonic paracellular permeability, and adapting cardiomyocyte to hemodynamic overload. Moreover, our findings suggest a novel role of MACF1 in bone tissue. More importantly, mutation in MACF1 has been described in a novel myopathy disease in humans and cancer development. Thus, MACF1 is attracting more attention.
Although MACF1's functions have been extensively studied and uncovered, many aspects of MACF1 have not been well understood. For example, how is the MACF1 expression regulated? What is the role of MACF1 with its ATPase activity? What is the exact role of MACF1 in cancer development? Considering the fact that MACF1 is involved in both osteoblast function and Wnt/-catenin signal transduction critical for the regulation of osteoblast differentiation and bone formation, it will be interesting to see whether MACF1 can control bone formation via Wnt/-catenin signaling. Further study of MACF1 is merited to unravel interesting and exciting new functions of MACF1 in both physiological and pathological settings.


Conversion of C2-C4 Low Alkanes on ZSM-5 Containing Zn Nanoparticles

Mixed catalysts were prepared on the basis of a synthesized high-silica ZSM-5 zeolite and an electroexplosion nanosized zinc powder. The influence of Zn concentration in a zeolite on its acidic and catalytic properties in the reaction of C2-C4 low alkanes aromatization as well as the catalyst deactivation resulting from carbonization were studied. Eurasian ChemTech Journal 3 (2001) 1-9  2001 al-Farabi Kazakh St.Nat. University Introduction Catalysis of chemical reactions is an important and wide field of application of metal nanosized particles [1-3]. A high catalytic activity of these particles is explained by electron and geometric effects resulting from a small particle size [4-6]. A high dependence of the catalytic activity on the particle size emphasizes the need to develop selective methods to produce nanosized particles with a precision of 1-2 atoms. One of the rapidly developing methods for the synthesis of nanocrystal powders is the conductor electroexplosion carried out by passing a powerful current pulse with a width of 10-10 A/mm [7,8]. As a result, the formation of energetic and geometric structures occurs at extremely high temperature gradients that leads to an insignificant non-equilibrium of physicochemical processes. Metastable particles formed are characterized by structure deficiency, a developed inner surface and a high energy saturation. Obviously, this complex of the properties of nanosized powders (NSP) produced by electroexplosion determines their high catalytic activity in chemical processes. In this connection the study of physicochemical properties of nanosized metal powders and establishment of the catalysis fields where they would find a practical application is a very important and urgent task.

Introduction
Catalysis of chemical reactions is an important and wide field of application of metal nanosized particles [1][2][3]. A high catalytic activity of these particles is explained by electron and geometric effects resulting from a small particle size [4][5][6]. A high dependence of the catalytic activity on the particle size emphasizes the need to develop selective methods to produce nanosized particles with a precision of 1-2 atoms. One of the rapidly developing methods for the synthesis of nanocrystal powders is the conductor electroexplosion carried out by passing a powerful current pulse with a width of 10 4 -10 6 A/mm 2 [7,8]. As a result, the formation of energetic and geometric structures occurs at extremely high temperature gradients that leads to an insignificant non-equilibrium of physicochemical processes. Metastable particles formed are characterized by structure deficiency, a developed inner surface and a high energy saturation. Obviously, this complex of the properties of nanosized powders (NSP) produced by electroexplosion determines their high catalytic activity in chemical processes. In this connection the study of physicochemical properties of nanosized metal powders and establishment of the catalysis fields where they would find a practical application is a very important and urgent task.

Experimental
The synthesis of ZSM-5 with a silicate modulus SiO 2 /Al 2 O 3 =50 was carried out by hydrothermal crystallization of alkali alumosilicagels at 175°C for 4-6 days. A 30% sol of silica acid or water glass (29% SiO 2 , 9% Na 2 O, 62% H 2 O) was the silicon source, aluminum nitric salt was aluminum oxide source. The initial reaction mixture had the composition as follows: 17.5 Na 2 O·6.1R·Al 2 O 3 ·60 SiO 2 ·3275 H 2 O, where R is hexamethylenediamine. After the crystallization the zeolites were washed by the distilled water and dried at 100°C for 24 hours. To change the samples over the active H-form they were twice decationized by 25 % aqueous NH 4 Cl solution followed by drying at 110°C and calcined on air at 540°C for 6 hours. (Na 2 O content in Н-ZSM-5 was less than 0.02%).
The quality of the samples produced was checked by infrared spectroscopy (IRS), electron microscopy and X-ray structure analysis (XSA). Microscopic studies were carried out on SEM LEO-420 microscope at the resolution of 15 Å. In accordance with IRS (SPECORD M80) and XSA (DRON-3 and CuK α irradiation) data the samples synthesized have a high cristallinity degree (96%), do not contain amorphous phase admixtures and are qualified as ZSM-5 type.
A nanosized Zn powder was produced on a laboratory installation by the electroexplosion of a wire 0.1-0.2 mm in diameter in argon atmosphere. Wire electroexplosion is a drastic change in the physical state of a metal due to an intensive energy release when passing a high-density pulsed current [9]. On the condensation in the flow of a rapidly broadening vapor a zinc powder was produced with an average particle sized of approximately 100 nm which had a spherical shape. Using a ball vibrating mill the samples having different contents of the promoter were pre-pared by dry mixing of a zeolite and Zn NSP on air for 2 hours. To carry out comparative studies we prepared zeolite catalysts where the commercial zinc was introduced by ion exchange or mechanical mixing. Catalyst characteristics and their codes are given in Table 1. The catalysts were pressed into pellets. A fraction of 0.25-0.50 mm was taken for tests using special sieves.
hydrocarbons. The tests were carried out on the flow type installation, with the reactor volume of 5 cm 3 at atmospheric pressure, reaction temperature of 350-600°C and volumetric flow rate of 50-400 h -1 . The reaction products were analyzed by gas chromatography. The stability of the catalyst operation was assessed by the changes in the degree of the initial raw material conversion and in the selectivity of the formation of aromatic hydrocarbons.
DTA-DGA patterns of carbided catalysts were recorded using «C» DTA-DGA instrument (MOM, Hungary) within 20-1000°C. The sample (400-500 mg) was heated in a platinum crucible on air at the rate of 10 grad /min. Table 2 shows the results of the investigations of acidic properties of zeolites modified by Zn nanosized powders.

Results and Discussion
An unmodified zeolite has two types of acidic centers that is evidenced by the presence of two forms of ammonia desorption on a TD spectrum: weak acidic sites with the temperature of the peak (T max ) of 210°C and strong acidic sites with T max of 425°C. The concentration of weak acidic sites is 270.5µmol/g, that of strong acidic sites is 243.7 µmol/g. Activation energy is 33.3 and 123.3 kJ/mol for I and II forms of ammonia desorption, respectively. When adding a small amount of Zn NSP to a zeolite, the strength and the concentration of strong acidic sites decrease. When zinc concentration is brought to 4%, the smoothening of the high-temperature maximum on the thermodesorption spectrum is observed and its exact determination becomes impossible. In addition, the strength and the concentration of weak acidic sites as well as the activation energy of ammonia desorption decrease (Table 2). Therefore, the investigations of acidic properties of a catalyst demonstrated that on the introduction of Zn NSP into a zeolite the redistribution of acidic sites by strength and concentration as well as the formation of new acidic sites with a higher energy of ammonia retention occur. The most significant influence is caused by Zn NSP on strong acidic sites of a zeolite; a pronounced decrease in strength and concentration of weak acidic sites is observed at the zinc content more than 1%. As a result the ratio between weak and strong acidic sites of a zeolite changes that influences its catalytic properties and hydrocarbon conversion because specific active sites are responsible for the oc-  The testing of acidic properties of the catalyst surface was carried out by temperature-programmed desorption (TPD) of ammonia. The procedure of TPD experiments has been described in [10]. To calculate the values of activation energy, ammonia desorption from the samples was carried out at different heating rates -5, 10, 15 and 20 grad/min. Activation energies were determined in accordance with the procedure described in [11].
The catalytic activity and stability of zinccontaining zeolites were studied in the process of the conversion of C 2 -C 4 alkanes mixture into aromatic 3 currence of one or other chemical reaction.
In the presence of a novel class of heterogeneous catalysts -ZSM-5 high-silica zeolites -C 2 -C 4 saturated hydrocarbons are converted into a mixture of C 6 -C 9 aromatic hydrocarbons (mainly, benzene, toluene, xylenes) and higher that may be used as highoctane additives to motor fuels or as individual components after rectification. Gaseous products contain hydrogen, methane, and C 2 -C 4 hydrocarbons of different homologous series. The selectivity of aromatic hydrocarbon formation on the initial ZSM-5 is usually about 30% at the process temperature of 550-600°C. It is possible to improve the catalyst selectivity toward the formation of aromatic hydrocarbons by introducing promoters. Zn belongs to such promoters; in this case the concentration of the metal introduced determines the catalyst activity.
Preliminary tests of the catalytic activity of Zn NSP in the process studied have shown that the feedstock conversion does not occur under the above operation conditions. The rise in the temperature above 600°C leads to the occurrence of the reactions of thermal cracking. It is connected with the fact that alkanes conversion occur only in the presence of a bifunctional catalyst with an optimum combination of dehydrogenating and acidic properties. Fig. 1 shows the results of the study of C 2 -C 4 alkanes on a modified zeolite catalyst. On the introduction of 0.125 wt% of Zn NSP into a zeolite an increase in the total catalyst activity (evaluated by the degree of the feedstock conversion) is observed at 500°C, at 550°C the initial mixture of C 2 -C 4 n-al-kanes is conversed almost completely. On an unmodified zeolite the conversion degree reaches 99% at 600°C only. At 500-550°C the content of aromatic hydrocarbons increases, the concentration of С 1 -С 2 alkanes in the reaction products decreases. The increase in the volumetric flow rate by 2 times leads to a small decrease in the conversion degree, whereas the selectivity of aromatic hydrocarbon formation increases by more than 3%. The selectivity of hydrogen and С 2 -С 3 olefins formation also increases. When Zn NSP concentration in a zeolite increases to 0.25 wt%, the catalyst activity in the reaction of low alkanes aromatization further increases. Thus, the selectivity of arenes formation on C-2 catalyst at 550°C and 100 h -1 was 49.7% at the conversion degree of Table 2 Acidic properties of the initial zeolite and a zeolite containing different amounts of Zn NSP 99%, whereas on C sample these characteristics were 28.5 and 96%, respectively. Zn NSP concentration in a zeolite brought to 0.5 wt% led to a still more significant increase in the aromatizing catalyst activity, the selectivity of arenes formation was 51.3% at 550°C, that is almost by 2 times higher than on the initial catalysts. At the same time decreases the formation of by-products (С 1 -С 2 alkanes). The increase in the volumetric flow rate to 200 and 300 h -1 leads to a smooth decrease in the conversion degree and the selectivity of arenes formation and to the increase in the selectivity of С 1 -С 2 alkanes and С 2 -С 3 olefins formation. It should be also noted that despite of the increase in the volumetric charge on the catalyst by 2-3 times its aromatizing activity remains high and the selectivity of arenes formation exceeds 40%. Zn NSP concentration brought to 0.75 wt% has led to the decrease in the selectivity of aromatic hydrocarbons formation as compared to C-3 catalyst by 8.6% at 550°C and 100 h -1 , although total catalyst activity retains high (the conversion degree is 98%). At the further increase in Zn NSP concentration in a zeolite by 0.25 wt%, i.e. when its content is brought to 1 wt%, a more significant decrease in the selectivity of aromatic hydrocarbon formation and the increase in the formation of by-products occur. On the increase in Zn NSP concentration in ZSM-5 the general tendency of decrease in the selectivity of arenes formation and increase in С 1 -С 2 alkanes formation retains. The catalysts containing 1, 2, and 3 wt% of Zn NSP exhibit a high aromatizing activity at the initial process temperatures, i.e. at 450-500°C. There is observed a formation of a rather large quantity of aromatic hydrocarbons. When the temperature rises to 550°C, there is no rise in arenes yield on these catalysts, by the contrast a large amount of by-products is formed. It should be noted that on C-6 and C-7 catalysts practically the same amount of aromatic hydrocarbons is formed as on the initial catalyst.
Thus, the results obtained evidence that the increase in Zn NSP content in ZSM-5 to 1 wt% and higher leads to a sufficient decrease in the catalyst activity in the reaction of С 2 -С 4 low alkanes aromatization and to the increase in its activity in cracking. The optimum Zn NSP concentration in a zeolite is 0.5 wt% because on C-3 catalyst the largest quantity of aromatic hydrocarbons is formed (51.3%). Table 3 shows the data on the influence of Zn NSP concentration in a zeolite and the nature of the modifier on the yield of the desired product and the composition of gaseous and liquid products of С 2 -С 4 low alkanes conversion at 550°C and 100 h -1 . The yield of aromatic hydrocarbons sufficiently increases at the zeolite modification by Zn. Thus, on an unmodified ZSM-5 the yield was 27.4%, whereas on C-1 catalyst it was already 34.1 wt%. The yield reaches 49-50% on zeolites containing 0.25 and 0.50 wt%. The increase in Zn NSP concentration in ZSM-5 to 0.75 wt% and higher leads to the decrease in the yield of the catalysate that contains less С 6 -С 8 aromatic hydrocarbons, the fraction of С 9+ hydrocarbons being drastically increased. With the increase in Zn NSP content in a zeolite from 1.0 to 3.0 wt% the gaseous products contain more methane and less ethane and hydrogen, olefins content practically does not change. An insignificant content of С 2 -С 3 olefins evidences that they are intermediate products that actively enter further interactions leading to the production of aromatic hydrocarbons. Thus, by analyzing the data on the influence of Zn NSP concentration in a zeolite on the composition of the conversion products of the mixture of С 2 -С 4 low alkanes, it is possible to note that with the increase in the concentration the contents of benzene, toluene, xylenes in the reaction products change distinctly and pass through the extremum, the concentration of higher molecular aromatic compounds passes through minimum. The maximum quantity of benzene -toluene -xylenes fraction (BTXfraction -82.7%) is in the liquid product obtained on C-3 catalyst.
As seen from the data of the Table 3, quantitative compositions of the products obtained on the catalysts with different kinds of Zn differ greatly. Thus, the largest quantity of BTX-fraction is formed on C-3 and C-11, in this case the compositions of the liquid products obtained are close. So is the composition of the catalysts obtained on C-8 and C-9 samples. Hydrogen, ethylene and propylene contents in gaseous products obtained on different Zn-containing catalysts vary insignificantly, whereas methane and ethane contents differ greatly: a larger amount of methane and that smaller of ethane are formed on C-3 and C-10 catalysts than on C-8, C-9 and C-11 catalysts. The differences revealed in the composition of gaseous and liquid products formed on the catalysts under study are conditioned by different rates of the occurrence of individual process stages, i.e. the properties of a catalyst depend on the method for the introduction of the modifying metal, its physicochemical properties and the electron state of the catalyst.
On the basis of the experimental data obtained from the study of acidic and catalytic properties of the

Table 3
Composition of the products of C 2 -C 4 low alkanes conversion on a zeolite catalyst containing different amounts of Zn NSP, and on the conventional catalysts (T reaction =550 °C, W=100 h -1 ) samples prepared it is possible to establish the role of the specific acidic sites in the acceleration of individual stages of the process of low alkanes aromatization. Thus, the activity of the initial H-ZSM-5 is determined by the presence of strong Brönsted sites where take place the cracking of saturated hydrocarbons and further transformations of olefins formed (oligomerization, hydrogen redistribution etc.). For Zn/HZSM-5, the dehydrogenation of initial paraffins and aromatization of unsaturated compounds occurs over strong Lewis sites containing Zn 2+ cations. That is why, to obtain an effective catalyst of С 2 -С 4 low alkanes aromatization, an optimum proportion of strong Lewis and Brönsted acidic sites is necessary. The fraction of strong Lewis sites being determined by Zn NSP concentration in HZSM-5, to reach an optimum proportion of acidic sites of different types, this concentration should be 0.5 wt%, as shown above.
As seen from the data of Table 4, the activity of conventionally produced C-8, C-9 and C-10 catalysts compares well with the activity of C-3 catalyst. The degree of conversion of the initial feedstock retains high (98-99% at 550°C and 100 h -1 ). Nevertheless, the selectivity of aromatic hydrocarbon formation on C-8, C-9 and C-10 catalysts is sufficiently lower than on C-3 catalyst. A zeolite where zinc was introduced by ion exchange followed by the impregnation (43.7%) has the highest selectivity towards aromatic hydrocarbons. The formation of by-products (С 1 -С 2 alkanes) increases on the catalysts produced using conventional methods that evidences a significant reaction rate of cracking and hydrogenation of intermediate compounds formed.
The application of C-11 catalyst allows producing aromatic hydrocarbons from saturated С 2 -С 4 hydrocarbons at 450°C and 100 h -1 . With the increase in the reaction temperature a significant increase in the conversion degree and selectivity of arenes formation occurs. At 550°C and 100 h -1 these values are 96 и 46.3%, respectively. The activity of C-11 catalyst is lower than that of C, C-8, C-9 and C-10 catalysts, its selectivity for aromatic hydrocarbon formation is somewhat higher. Nevertheless, its total and aromatizing activities are lower than those of C-3 catalyst are. Fig. 2 shows the results of the study of the influence of the operating period of Zn-containing catalysts on their catalytic properties. Since unmodified ZSM-5 reveals the highest activity in the reaction of low alkanes aromatization at 600°C, the determination of its stable operation period was carried out at this tem-perature. On Zn-containing zeolites the initial gaseous feedstock is conversed almost completely at 550°C, that is why the period of their time between regenerations was determined at that temperature.
The initial Н-ZSM-5 retains its catalytic activity constant for about 24 hours, then the decrease in the conversion degree and in the selectivity of the formation of aromatic hydrocarbons is observed. At the same time the increase in the selectivity of low olefins formation occurs, and the selectivity of alkanes formation changes insignificantly.
Zn NSP introduction into a zeolite leads, along with a significant increase in the catalyst aromatizing activity, to the rise in its operating period. Zeolites containing Zn NSP or conventional catalysts have a stable operating period for 40 hours. A commercial catalyst has a stable operating period of 8 hours, then a drastic decrease both in the conversion degree of the initial feedstock and in the yield of aromatic hydrocarbons are observed. At the same time the concentration of C 2 -C 3 olefins rises being connected with the deceleration in the reaction rate of hydrogen redistribution.
Conventional catalysts (C-8 and C-10) are characterized by very close indices of activity and stability in the process of low alkanes aromatization. A zeolite containing 0.5% Zn NSP possesses the highest operation stability among the samples studied. We also should note a high catalytic operation stability of C-2 sample. In 40 hours of continuous operation the conversion degree and selectivity of aromatic hydrocarbon formation on C-3 decrease only by 5 and 3.8% respectively, in 64 hours -by 19 and 6.7%. On conventional catalysts the decrease in these indices in 64 hours of operation is 25-28% and 8.2-10.7%. In addition, the yield of aromatic hydrocarbons formed on C-3 sample during all the operating period is significantly higher than on other catalysts. Thus, from the above it appears that C-3 catalyst is the most effective in С 2 -С 4 low alkanes aromatization. Its activity, selectivity and stability are significantly higher than those of traditional catalysts and commercial samples. The selectivity of aromatic hydrocarbon formation on C-3 exceeds 50%, the conversion degree being 99% at the reaction temperature of 550°C and the volumetric feedstock flow rate being 100 h -1 . The time between regenerations is more than 60 hours, but the catalyst keeps a rather high catalytic activity towards the desired products after this period of time, which allows continuing the process if necessary. To establish the optimum duration Table 4 Characteristics of the products of the conversion of C 2 -C 4 low alkanes mixture on ZSM-5 and Zn/ZSM-5 Note. W is the volumetric rate; X is the conversion degree; S 1 , S 2 , S 3 and S 4 are the selectivities of the formation of hydrogen, C 1 -C 2 alkanes, C 2 -C 3 alkenes and aromatic hydrocarbons, respectively; А is the yield of aromatic hydrocarbons. of the reaction cycle of a catalyst after which there would be no problems connected with the restoration of its catalytic activity by oxidative regeneration it is very important to study the process of coke formation and the nature of coke deposits.
In accordance with the data of differential thermal analysis for all the samples studied, the presence of overlapping exothermal effects within 460-920°C with pronounced extreme points within 620-640°C and less pronounced extremes points in the high-temperature region was established. This evidences the presence of a wide range of coke deposits (Fig.3).
to a higher-temperature region, especially for C-10 catalyst.
Thus, the data on differential thermal analysis show that the introduction of Zn NSP into a zeolite leads to the decrease in the intensity of coke formation and production of less condensed coke deposits that make possible a relatively easy oxidative regeneration. The data obtained agree well with the results of the tests of the catalytic stability of Zn-containing catalysts. For example, C-3 catalyst reveals the highest activity in the process of C 2 -C 4 low alkanes aromatization that is due to the decrease in the reaction rate of coke formation on this catalyst.

Conclusions
By mechanical mixing produced were zeolite catalysts containing different amounts of the electroexplosion nanosized Zn powder and conventional Zn as well the zeolites where Zn was introduced from Zn nitrate solution by ion exchange followed by impregnation. Zn introduction into a zeolite leads to the formation of strong aproton acidic Zn 2+ -containing sites characterized by a high energy of ammonia retention. The zeolites with Zn NSP introduced possess higher catalytic activity and stability in the aromatization of C 2 -C 4 low alkanes as compared to conventional zinc-containing catalysts. A catalyst containing 0.5 wt% Zn NSP produced by mechanical mixing of Zn powder and ZSM-5 zeolite is the most effective for this process. The largest amount of coke is contained on the sample C (19.3%) that is explained by an intensive coke-formation reaction. Smearing of peaks and higher burn-out temperatures of coke deposits are observed on DTA curve for the catalyst C as compared to Zncontaining catalysts that points to their higher polycondensation degree. That is why the regeneration of this catalyst will be extremely difficult. Zn-containing catalysts have a lower quantity of coke deposits, the compaction products formed are less condensed that is confirmed by a lower temperature of the end of the coke burning.
The least coke amount is on C-3 catalyst (11.1%), this catalyst is characterized by the presence of an exothermal peak in the region from 460 to 680°C with a clear maximum at 620°C and the II form in the shape of a small shoulder before 860°C. In 64 hours of the operation of C-2 and C-10 catalysts larger amounts of coke were formed (11.7 and 14.3%, respectively), the form II and the end of the coke burnout are shifted


extfieldsofstudy: Chemistry Medicine

text:
Development of portable defocusing micro-scale spatially offset Raman spectroscopy

We present, for the first time, portable defocusing micro-Spatially Offset Raman Spectroscopy (microSORS). Micro-SORS is a concept permitting the analysis of thin, highly turbid stratified layers beyond the reach of conventional Raman microscopy. The technique is applicable to the analysis of painted layers in cultural heritage (panels, canvases and mural paintings, painted statues and decorated objects in general) as well as in many other areas including polymer, biological and biomedical applications, catalytic and forensics sciences where highly turbid stratified layers are present and where invasive analysis is undesirable or impossible. So far the technique has been demonstrated only on benchtop Raman microscopes precluding the non-invasive analysis of larger samples and samples in situ. The new set-up is characterised conceptually on a range of artificially assembled two-layer systems demonstrating its benefits and performance across several application areas. These included stratified polymer sample, pharmaceutical tablet and layered paint samples. The same samples were also analysed by a high performance (non-portable) benchtop Raman microscope to provide benchmarking against our earlier research. The realisation of the vision of delivering portability to micro-SORS has a transformative potential spanning across multiple disciplines as it fully unlocks, for the first time, the non-invasive and non-destructive aspects of micro-SORS enabling it to be applied also to large and non-portable samples in situ without recourse to removing samples, or their fragments, for laboratory analysis on benchtop Raman microscopes.

Introduction
Non-invasive analysis of thin stratified turbid (diffusely scattering) layers is an analytical challenge commonly present in a wide range of disciplines. For instance, many analytical problems in art require non-invasive probing of thin stratified layers of paint that may lie beyond the reach of conventional methods such as confocal Raman microscopy. The sublayer chemical information is crucially important, for example, in restoration and conservation processes or to elucidate technique of the artist in question. The key goal in all these applications is to avoid cross sectional analysis which is either highly undesirable or not permissible due to the uniqueness and high cultural value of the object analysed. Examples of such matrices in cultural heritage include artworks such as panels, canvases and mural paintings, painted statues and other decorated objects in art. Painted layers, in general, are by their nature highly turbid and typically only a few tens of micrometres thick, commonly spreading in multiple stratigraphy leaving them often out of reach of conventional Raman microscopy, which is frequently the technique of choice in cross-sectional analysis, where permitted. 1,2 This is only one area on which we are exemplifying the analytical need but there are many other disciplines having similar requirements including polymer, catalytic, biological and biomedical applications and forensic sciences where highly turbid stratified layers can be present and where invasive analysis may be undesirable or impossible.
Recently a new concept in Raman microscopy possessing considerably higher penetration depth than that of conventional Raman microscopy and capable of overcoming the above limitations in a number of situations has emergedmicro-SORS. 1,2 The method builds on earlier advances of its parent technique, (macro-scale) Spatially Offset Raman Spectroscopy (SORS) 3 and relies on diffusion properties of photons in turbid media. The most basic variant of micro-SORS is defocusing micro-SORS. 1 Although this is not the most effective micro-SORS concept in existence, 4 by not involving fully separated laser illumination and Raman collection zones as in classical SORS, it is undoubtedly the most practical as it can be practised on existing benchtop Raman microscopes without any modifications. 5,6 Nevertheless, to date the technique has only been demonstrated on benchtop Raman microscopes. As such the method has not delivered fully on its non-invasive and non-destructive potential as samples need to be mobile and of adequately small size in order to fit under a Raman microscope. Many applications in the abovementioned disciplines, however, do not fulfill these criteria. Taking again the example of cultural heritage where only small portable objects can be analysed in this manner unless a fragment of a large or non-portable art objects can be taken away, which by its nature breaches the requirement for non-invasiveness. The work presented here concerns the development of a transformative capability facilitating portability and permitting operation outside specialized laboratory. This was accomplished by converting an existing conventional commercial portable Raman instrument to micro-SORS by equipping it with a microscopic capability. This article describes the instrument design and characterises its performance on several examples of artificially assembled two-layer samples. The same samples have also been characterised on our existing micro-SORS Raman benchtop system to provide benchmarking against our previous research.
The concept of defocusing micro-SORS and implemented modifications are illustrated in Fig. 1. A conventional portable Raman instrument was fitted with a microscope objective. In general, the basic micro-SORS measurement of a two layer turbid sample relies on collecting at least two Raman spectra: (i) the first spectrum is acquired with the sample at a standard 'imaged' position and corresponds to a standard Raman microscopy surface Raman measurement, (ii) the second measurement is taken with the sample moved away from the microscope objective by a 'defocusing distance Δz' to a 'defocused position'. The effect of sample displacement is the enlargement ('defocusing') of both the laser illumination and Raman collection zones on sample surface. 5 The larger the defocusing distance Δz the greater the enlargement of both the laser illumination and Raman collection zones on sample surface. The 'imaged' position measurement typically yields a Raman spectrum dominated by the surface layer 1 and would correspond conceptually to a zero-spatially offset measurement in conventional SORS analysis. The measurement carried out in the 'defocused' position produces a Raman spectrum which has a significantly higher degree of Raman contribution from sublayers relative to the surface layer with stratified turbid samples, in analogy with a non-zero spatially offset spectrum acquired in a conventional SORS measurement. That is the contrast of the sublayer improves (although the overall Raman intensity of both the layers diminishes). The relative intensity change within spectral subcomponents signifies the presence of multiple layers in sample and permits the recovery of pure Raman components of individual layers.
In the case of a two layer sample, only a very simple mathematical manipulation of the 'imaged' and 'defocused' spectra can then be performed to retrieve a pure Raman spectrum of sublayer. This process comprises a scaled subtraction of the 'imaged' spectrum from the 'defocused' spectrum cancelling the contribution of the top layer. More defocused spectra need to be acquired for the separation of layers in a system consisting of more than 2 layers. 5 The concept of scaled subtraction was first applied to confocal Raman microscopy with transparent samples by the Morris group. 7,8 Micro-SORS uses similar approaches but instead of direct imaging it utilises diffuse component of light.
In general, the limitations of the micro-SORS technique include inapplicability to highly absorbing, extremely thin sublayers, compounds with low Raman cross sections at the subsurface position and highly fluorescing samples. In addition, samples possessing very high heterogeneity across their surface or within sublayers are also challenging without recourse to a more complex data acquisition methodology. 2 It should also be noted that the spatial resolution of micro-SORS is, in general, significantly inferior to that of conventional Raman microscopy as it relies on diffused component of light and as such it should only be used at depths where conventional confocal Raman microscopy is inapplicable. Surface roughness can however be dealt with as the roughness will manifest itself as a variation of Raman signal intensities between different layers and as such it will subtract away in the scaled subtraction when retrieving individual layer signals. The effect could however confuse the interpretation of the order in which layers are laid derived typically from the signal dependencies on the degree of defocusing. 4 A study by Conti et al. 9 further contrasts the conventional confocal Raman microscopy with micro-SORS pointing at differences and commonalities between the two techniques and underlying processes.

Portable defocusing micro-SORS
The measurements were carried out using a portable Xantus-2™ (Rigaku, Boston, USA) Raman spectrometer with 2000 × 256 pixels, thermoelectrically cooled CCD and a selected excitation wavelengths of 785 nm. The instrument was modified by removing the end collection and focusing lens with its tubular housing and replacing it with a standard microscope 20× objective (WD 1.3 mm, NA 0.4, Olympus) placed in contact with the front cover plate of the instrument. No other modifications were implemented. This step was important equipping the instrument with micro-scale sampling capability and enabling it to perform effective micro-SORS measurements.
The micro-SORS spectra were acquired with a laser power at the sample of up to nominal 250 mW and a spectral resolution of 7-10 cm −1 . Both the 'imaged' and 'defocused' spectra were acquired with acquisition times ranging from 3 to 5 s with 7 accumulations used (i.e. the total acquisition time per sample position was 21-35 s). The instrument was operated from an external computer (laptop). Combined sample illumination and collection area of the modified instrument was estimated to be approximately 40 µm in diameter by scanning across an edge of sample. In this measurement a sharp edge of sample was moved across the interaction zone perpendicular to the optical axis of the instrument. The Raman intensity of the surface layer was recorded as a function of position of sample. The diameter of the interaction area was estimated as the separation of the 10 to 90% intensity points.
Except for the painted layer system, raw spectra are presented with no background correction; all the spectra were normalized to a Raman band of respective top layer. The subtracted pure Raman spectra of individual layers were rescaled relative to each other for clarity. The sample was placed on a manual micro-positioning stage to facilitate measurements at different distances from the objective. In analogous way, the portable instrument could, to an equal effect, also be located itself on a micro-positioning stage and moved in front of sample permitting the analysis of larger, non-moveable objects.

Benchtop defocusing micro-SORS
The measurements were carried out using a Senterra dispersive micro-Raman spectrometer (Bruker Optik GmbH) that includes a standard confocal optical microscope (Olympus BX51). The Raman spectra were acquired using the same objective as that used in the portable instrument (20×, WD 1.3 mm, NA 0.4) and a laser 785 nm excitation wavelength with a nominal power of 100 mW. The detection system consisted of a Peltier cooled CCD detector (1024 × 256 pixels), 1200 grooves per mm grating and the largest confocal slit (50 µm × 1000 µm) was used. Its spectral resolution was 3-5 cm −1 . The spectra were acquired with total acquisition time of 5 s and accumulations ranging from 5 to 25 (i.e. the total acquisition time per sample position was 25-125 s). Combined sample illumination and collection area with the 20× objective was estimated to be approximately 4 µm in diameter by scanning across an edge of sample as above.
Except for painted layer system, raw Raman spectra are presented with no background correction. All the spectra are normalized to a Raman band of respective top layer. The subtracted spectra are rescaled relative to each other for clarity.

Specimens
Multiple layers of different polymers are used in a number of fields, for example in food packaging. This application area is conceptually represented by our first specimen (S1) comprising artificially assembled two-layer polymeric system. Both layers were highly turbid and opaque in appearance, like many polymer materials present in our daily lives. The top layer was a polytetrafluoroethylene (PTFE) tape (75 µm thick) placed over a 1 mm thick sublayer of a plastic spoon made of polystyrene (PS).
Many pharmaceutical products are prepared using different micrometric highly diffusely scattering layers. Our second specimen (S2) representing this area is a pharmaceutical tablet consisting of an external coating layer containing anatase (TiO 2 , 70 µm thick) followed by a 4 mm thick pharmaceutical formulation (naproxen acid and excipients).
The third example (S3) is an artificially prepared two-layer system simulating application of a real spray paint on stone: PY151 acrylic spray colorant was spread with a thickness of ∼50 µm on a marble stone (1 cm thick), consisting of calcite (CaCO 3 ).
The fourth example (S4) consists of painted layers simulating a real artistic stratigraphy: two pigments were used, red ochre (hematite -Fe 2 O 3 ) (60 µm thick) on top of phthalocyanine blue (C 32 H 16 N 8 Cu -50 µm thick), both deposited on a sheet of paper. The preparation of the last two specimens was suggested from the critical need in conservation of cultural heritage to obtain information on the composition of painted stratigraphy applied on panel, canvas or statue and on the conservation state of substrates in mural paintings (Table 1). Red ochre (60 µm) Phthalocyanine blue (50 µm) Fig. 2 Defocusing micro-SORS spectra of a stratified plastic sample (S1) acquired with portable (a) and benchtop (b) instruments. Defocusing distances in μm are indicated next to the spectra which are normalized to the PTFE band (top layer) at 731 cm −1 ; asterisks indicate PS bands. Subtracted spectra of sublayer derived numerically from micro-SORS measurements (c): spectra are obtained by subtracting the scaled "imaged" spectrum from the defocused one. Reference spectrum, acquired with benchtop instrument, is shown in red.

Results and discussion
The first demonstration of the performance of portable defocusing micro-SORS was carried out on a stratified plastic system (S1) composed of PS spoon covered with a PTFE tape. Fig. 2 shows the results of the measurements acquired both with portable and benchtop instruments. Raman spectra of the sample when sharply imaged ("0", zero position) yield the contribution of both layers, clearly visible through their characteristic and intense bands at 1001 cm −1 and 731 cm −1 for PS and PTFE, respectively. As the sample is displaced from the zero position, moved farther away from the microscope objective, the contribution from the sublayer relative to the top layer dramatically increases. To recover the pure Raman spectrum of the sub-layer, the zero spectrum was subtracted from the defocused one, cancelling the Raman contribution from the top layer (Fig. 2c). The resulting pure component of the bottom layer compares well with the reference spectrum of this layer, both with portable and benchtop instruments.
Portable defocusing micro-SORS method is illustrated on another example of a two layer system, coming from the pharmaceutical field. The tablet (S2) is composed of naproxen acid coated by a thin film of anatase. Selected Raman spectra from the zero to 1-2 mm defocused positions are shown in Fig. 3 where the evolution of the signals of individual layers with the defocused positions is evidenced. The two layers change the relative intensity with respect to each other, as can be seen by observing in particular the strong bands of naproxen acid at 748 and 524 cm −1 and the band of anatase at 639 cm −1 . By applying the same scaling method to the two raw spectra one is able to recover the estimates of the pure Raman sublayer component, as shown in Fig. 3c; the spectra of the sublayer fit again well with the reference spectrum, both for portable and benchtop measurements. The higher spectral resolution of the benchtop instrument turns out to be essential to discriminate the doublet at 396 cm −1 and at 406 cm −1 , belonging to anatase and naproxen acid respectively, which is confused in a single band with portable instrument. Fig. 3 Defocusing micro-SORS spectra of a coated pharmaceutical tablet (S2) acquired with portable (a) and benchtop (b) instruments. Defocusing distances in μm are indicated next to the spectra, which are normalized to the anatase band (top layer) at 639 cm −1 ; asterisks indicate naproxen acid bands. Subtracted spectra of sublayer derived numerically from micro-SORS measurements (c): spectra are obtained by subtracting the "imaged" spectrum from the defocused one. Reference spectrum, acquired with benchtop instrument, is shown in red.
The third specimen mimics a spray paint on mural subsurface; the artificial two-layer system (S3) consists of a deposition of a yellow colorant (in acrylic media) on marble surface. At the "imaged" position the spectrum is dominated by contribution from the top layer, yellow spray, while calcite, the main component of the bottom layer, is detectable only through a very small band at 1086 cm −1 (Fig. 4). Moving the sample farther away from the microscope objective accentuates the inner layer: the increase of calcite is unequivocal from the zero to 3 mm of defocused positions, both for portable and benchtop measurements. The Raman spectra of sublayer recovered from the measurements are shown in Fig. 4c. Again, good agreement is reached with the reference spectrum in both the measurements.
The final example is the demonstration of the applicability of portable defocusing micro-SORS to a painted stratigraphy. The artificial sample is composed of red ochre micrometric layer spread on top of a phthalocyanine blue layer; a paper sheet was used as substrate. The evolution of the signals with the defocused position is shown in Fig. 5. In line with expectations, at the "imaged" position the spectrum is dominated by contribution from the top layer, red ochre. At the same position, phthalocyanine blue bands are more intense in the measurement acquired with portable instrument than that with benchtop one, most likely due to the larger laser spot size. In fact, the larger the spot size the higher the contribution of the bottom layer in the initial, "imaged" position. As shown in Fig. 5, with the increase of the defocused distances with the portable system the contribution of phthalocyanine blue (sublayer) becomes larger, even though to a minor extent when compared with the same evolution observed with the benchtop instrument. Nevertheless, the substrate spectrum was successfully recovered from both with portable and benchtop measurements (Fig. 5c).
It should be noted that the original, unmodified portable Raman instrument ( prior to fitting it with microscope objective) was also amenable to micro-SORS analysis as its original collection lens ( f = 21 mm, N.A. 0.21) in part also acted as a Fig. 4 Defocusing micro-SORS spectra of spray paint on marble (S3) acquired with portable (a) and benchtop (b) instruments. Defocusing distances in μm are indicated next to the spectra, which are normalized to the spray band (top layer) at 1143 cm −1 ; asterisks indicate PY151 bands. Subtracted spectra of sublayer derived numerically from micro-SORS measurements (c): spectra are obtained by subtracting the "imaged" spectrum from the defocused one. Reference spectrum, acquired with benchtop instrument, is shown in red.
lower magnification microscope objective. Nevertheless its performance was lower than that of the modified instrument. The unmodified design exhibited 1.6-times and 2.3-times lower enhancement for marble with spray (S3) and painted stratigraphy sample (S4), respectively, compared with the modified instrument (i.e. instrument equipped with a microscope objective). Also the initial contrast (the ratio of subsurface to surface layer Raman signal intensities) at the imaged position between the sublayer and surface layer was 3.3-times and 2.0times smaller for marble with spray (S3) and painted stratigraphy sample (S4), respectively, for the unmodified instrument. The smaller enhancement was present due to the fact that initial contrast was lower for the unmodified instrument and also as it possessed a lower numerical aperture leading to smaller diameter change with axial displacement.
In conclusion, it is interesting to note that the performance of the portable micro-SORS instrument is broadly speaking as good as that of the benchtop Raman microscope micro-SORSboth in terms of data quality of the retrieved sublayer spectra as well as the magnitude of the achieved contrast improvement between the subsurface and surface layers. Although some variations depending on type of sample are present due to the different nature of the optical layer and the two experimental setups (for example, the spectral resolution of the portable instrument is lower to that of the benchtop system). This exceptionally positive outcome of the experiments bodes well for practical deployment of the instrument on real samples planned in the near future as the next phase of our systematic development programme of work.

Conclusions
A concept of portable non-invasive micro-SORS measurements to elucidate hidden stratigraphy was demonstrated conceptually for a number of artificially prepared samples exemplifying a range of potential applications across multiple disciplines. The portability unlocks fully the non-invasive and non-destructive potential of micro-SORS. Given the wide applicability of the technique and potential for further improve- Defocusing micro-SORS spectra of painted stratigraphy (S4) acquired with portable (a) and benchtop (b) instruments. Defocusing distances in μm are indicated next to the spectra, which are normalized to the top layer band at 286 cm −1 ; asterisks and circles indicate red ochre and blue phthalocyanine bands, respectively. Subtracted spectra of sublayer derived numerically from micro-SORS measurements (c): spectra are obtained by subtracting the "imaged" spectrum from the defocused one. Reference spectrum, acquired with benchtop instrument, is shown in red. ments by introducing fully separated illumination and collection micro-SORS modality 4 the concept is expected to find a number of practical application niches in a number of areas providing a beneficial complementary analytical capability to conventional Raman microscopy in situations where the accessible depths of conventional Raman microscopy are inadequate. Such areas include art, archaeology, forensics, catalytic research, biology and biomedical sciences.


extfieldsofstudy: Medicine Chemistry

text:
Inclusion complex of tamibarotene with hydroxypropyl-β-cyclodextrin: Preparation, characterization, in-vitro and in-vivo evaluation

Graphical AbstractUnlabelled image

Introduction
Tamibarotene, 4-[ (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2naphthalenyl) carbamoyl] benzoic acid (Am80, the formula shown in Fig. 1), is a synthetic retinoid drug mainly used for relapsed or refractory acute promyelocytic leukemia (APL). Tamibarotene can be optionally combined with retinoic acid receptor α [1]; this combination recovers the normal function of primary marrow cells (PML) and the retinoic acid receptor gene leukemia α (RARα) by releasing the fusion of PML/RARα gene, which promotes the differentiation of promyelocytic cells, so that a therapeutic effect for APL can be achieved. Compared with other retinoid used for the treatment of APL, Am80 has a higher efficacy, produces less adverse reactions [2], and hardly has any drug resistance [3]. In addition, Am80 is less reactive to light, heat, oxidation, and other physical and chemical factors. It is especially suitable for relapsed or refractory APL patients who are resistant to all trans retinoic acid (ATRA) [4]. In 2005, tamibarotene tablet was first approved for use against acute promyelocytic leukemia in Japan, with the trade name Amnolake and specifications of 2 mg. As deeper research, tamibarotene is being investigated for various disease, studies have shown that tamibarotene can be used for multiple myeloma [5], Crohn's disease [5], chronic obstructive pulmonary disease [6], liver cancer [7][8][9][10], solid tumors [5] and Alzheimer [3]. However, tamibarotene has a poor solubility; when administrated orally, the first-pass effect is obvious, the bioavailability is relatively low (50%-70%) and the absorption is poor. In addition, Am80 has no injection available because of its poor water solubility. Therefore, improving the solubility of Am80 and increasing its bioavailability for clinical use are problems need to be solved urgently.
Several methods can be used to enhance the solubility of insoluble drugs, such as the use of suitable solubilizers, making poorly soluble drugs into soluble prodrugs, the use of nano technology and inclusion technique, etc. Among them, inclusion technique can significantly improve the stability and solubility of insoluble drugs, so as to improve its bioavailability [3,5]. The preparation of an inclusion complex is simpler and requires less additive materials, making it more suitable for commercial production. A dozen cyclodextrin-containing drugs have already been approved and marketed [11]. Furthermore, inclusion complex can be used as an intermediate of various dosage forms. Therefore, this study intends to adopt the inclusion technology to prepare a water-soluble tamibarotene inclusion complex with improved bioavailability.
Cyclodextrin and its derivatives are widely used as pharmaceutical materials for preparing inclusion complexes due to its non-toxicity, high biodegradability and various other advantages [11]. They can be used to improve the water-solubility, chemical stability and bioavailability of insoluble drugs, as well as reducing drug toxicity [12]. Among three types of cyclodextrin, β-CD is the least soluble, making it the easiest to be crystallized from water. Thus, β-CD is the most commonly used cyclodextrin. HP-β-CD is the ether derivative of β-CD. Compared with β-CD, HP-β-CD is more soluble and less toxic [13]. When administered via injection, HP-β-CD has almost no stimulation to muscle and mucosal. It has been approved by the United States Food and Drug Administration (FDA) to be used as a drug solubilizing agent and penetration enhancer. Thus, HP-β-CD has great potential applied in pharmaceutical excipients [14].
The aim of this study was to prepare a tamibarotene hydroxypropyl-β-cyclodextrin inclusion complex (Am80-HPβ-CD) through a freeze-drying method so as to improve the solubility and bioavailability of tamibarotene. To observe the formation of the inclusion complex, Am80-HP-β-CD was characterized by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). Next, the in-vitro dissolution characteristics of Am80-HP-β-CD and Am80 were verified with a dissolution method. Furthermore, the pharmacokinetics characteristics and the improvements in bioavailability were verified through the in-vivo experiments performed on rats. These results suggest that HP-β-CD will be potentially useful in the delivery of water-insoluble anticancer agents such as tamibarotene.

Preparation of Am80 -HP-β-CD inclusion complex
The Am80-HP-β-CD inclusion complex was prepared via a freeze-drying method. The Am80 was first dissolved in ethanol (3 ml), and then dispersed the Am80 solution into HP-β-CD aqueous solution at the molar ratio of 1:1. The obtained suspension was then stirred continually for 4 h at room temperature. The ethanol was then entirely removed by rotary evaporation for 15 min at 65°C, −0.08 MPa and then 10 ml distilled water was added and the solution was filtrated with a 0.45 μm microporous membrane. The solution was then cooled to room temperature and freeze-dried for 24 h. Finally, the inclusion complex was obtained.

Differential scanning calorimetry (DSC). The thermal
characteristics of the Am80, HP-β-CD physical mixture and the Am80-HP-β-CD inclusion complex were determined using a differential scanning calorimeter (DSC 204, Netzsch, Germany). Samples were accurately weighed in an aluminum pan, with an empty aluminum pan used as reference. The samples were then heated over a temperature range of 35-500°C, at the heating rate of 10°C/min under flowing nitrogen gas.

Fourier transform infrared spectroscopy (FT-IR). The FT-
IR spectra of Am80, HP-β-CD, physical mixture, and the Am80-HP-β-CD inclusion complex were obtained using a Fourier transform infrared spectrometer (Nicolet 6700 FT-IR Spectrometer, USA). Samples were prepared as KBr disks. The spectra were recorded from 4000 cm −1 to 400 cm −1 with 64 scans at the resolution of 4 cm −1 .

Study of in-vitro dissolution
The study of in-vitro dissolution of Am80-HP-β-CD (equivalent to 2 mg of Am80) and Am80 (2 mg

Pharmacokinetic study. The study of in-vivo
bioavailability was carried out on six adult Wistar rats (female or male) weighing 200 ± 20 g. The rats used for this study were housed individually under normal conditions and fasted overnight before the experiment with free access to water. Six rats were randomly assigned into two groups. One group was given tamibarotene solution through intragastric administration, while the other was given Am80-HP-β-CD inclusion complexes, both with dosage of 20 mg/kg. At specified time intervals (0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24 h after intragastric administration), 300 μl blood samples was collected from jugular venous sinus. Then the blood samples were processed according to 2.2.4.2.

Data analysis and statistics.
The plasma concentration versus time curves of the Am80 solution and Am80-HPβ-CD inclusion complex were plotted. Pharmacokinetic parameters were calculated by DAS 2.0 software. The corresponding sampling time (tmax) and the maximum peak concentration (Cmax) were directly obtained from the curves.
The powder was white and smooth. It is also soluble in distilled water. The aqueous solution is quite stable, it can be kept at 4°C for at least one month.

Differential scanning calorimetry (DSC)
Change of sample temperature in the DSC test occurred due to a phase transition, or endothermic and exothermic effect of the samples. Generally speaking, the phase transition and dehydrogenation reduction will produce an endothermic effect of reducing dehydrogenation, while crystallization and oxidation will produce an exothermic effect. As shown in Fig. 3, tamibarotene has a typically sharp endothermic peak at 231°C, HP-β-CD has a large characteristic absorption peak at about 360°C; the absorption peak of the physical mixture of HP-β-CD and tamibarotene is slightly different from that of the superimposed peaks of tamibarotene and HP-β-CD. The interactions of Am80 and HP-β-CD may potentially be the causes. The Am80-HP-β-CD inclusion complex has formed a new absorption peak at 320°C, without the typical peaks at around 231°C (Am80) and 360°C (HP-β-CD). This is caused by significantly weakened crystallization degree of tamibarotene when it formed an inclusion complex with HP-β-CD, which stops the thermal analysis from detecting the endothermic peak of the tamibarotene crystals (231°C). Comparing the absorption peak of tamibarotene (231°C) and HP-β-CD (360°C), we can conclude that the Am80-HP-β-CD inclusion complex has been synthesized successfully.

Fourier transform infrared spectroscopy (FT-IR)
The FT-IR spectra of Am80, HP-β-CD, Am80-HP-β-CD inclusion complex, and their physical mixture are shown in Fig. 4. These changes are relatable to HP-β-CD cavities of similar size, benzene, and cyclohexane group of Am80, inferring that the benzene and cyclohexane group of Am80 entered the HP-β-CD cavity, causing Am80 to be entrapped in the cavity of HP-β-CD.

In vitro dissolution study
The dissolution profiles of the Am80 solution and Am80-HPβ-CD inclusion complex were shown in Fig. 5. The cumulative dissolution percentage of Am80-HP-β-CD inclusion complex was  Fig. 4 -Spectrum of A(Am80), B (HP-β-CD), C (Am80-HP-β-CD) and D (the physical mixture).
significantly higher than that of Am80. As for Am80-HP-β-CD, the cumulative dissolution percentage was over 97% in the first 3 min, and stayed stable after 3 min, while for the Am80, the cumulative dissolution percentage was only 4.8% in the first 3 min, after 45 min, the cumulative dissolution percentage reached about 60% and stayed stable. The dissolution speed and the dissolution percentage of Am80-HP-β-CD were significantly higher than that of Am80. Thus, it can be concluded that the inclusion complex can significantly improve the dissolution efficiency. The cause could be that the Am80 crystal disappeared after the formation of Am80-HP-β-CD inclusion complex.

Analysis method validation results
The typical chromatograms of Am80, blank plasma and plasma contained Am80 were shown in Fig. 6. The retention time for Am80 was about 7 min. It was shown that the endogenous components of plasma have no interference with Am80. The standard curve showed a good linearity within the range of 0.1~9.6 μg/ml. Typical mean linear equation of standard curves for Am80 was A = 26.352C-0.5541, r = 0.999, where A represented the peak area of Am80 and C was the concentration of Am80. The RSD values of inter-day and intra-day precision at three concentrations were less than 5% and 10%. The RSD values of the extraction yields of Am80 at three different concentration levels were less than 15%. The results showed that the method had good accuracy and precision. The extraction recovery would satisfy the requirements of the pharmacokinetic study.

Pharmacokinetics and bioavailability study
Mean plasma concentration versus time profiles of Am80-HPβ-CD inclusion complex and Am80 were illustrated in Fig. 7. Pharmacokinetic parameters were calculated by using the DAS 2.0 software; the results were shown in Table 1. By comparing the results of the two formulations, Am80-HP-β-CD inclusion    To be specific, the AUC0-∞ of Am80-HP-β-CD inclusion complex was about 3.79 times compared with that of the Am80 after administration. Meanwhile, the Cmax of Am80-HP-β-CD inclusion complex was about 5.37 times compared with the Am80. The increase of AUC0-∞ and Cmax indicated the improvement of bioavailability. Evidently, the relative oral bioavailability of Am80-HP-β-CD inclusion complex increased about 2.79 times compared with Am80. Moreover, the tmax of Am80-HP-β-CD decreased 7 times compared with Am80, the decrease of tmax indicated Am80 can be absorbed into blood faster, which may be caused by the increase of solubility after the formation of the inclusion complex. The result of the in vivo studies was consistent with the in vitro studies. These results proved that the Am80-HP-β-CD inclusion complex could significantly enhance the oral bioavailability of various medications.
The poor absorption of Am80 may result from its low solubility and dissolution rate. The improvement of oral bioavailability for Am80 could be ascribed to the increase of its dissolution and water-solubility after Am80 entrapped into the HP-β-CD cavity.

Conclusion
In this study, the Am80-HP-β-CD inclusion complex was successfully prepared through a freeze-drying method at the mole ratio of 1:1. The DSC and FT-IR characteristics have proved the formation of the Am80-HP-β-CD inclusion complex. The invitro dissolution showed the dissolution efficiency of Am80-HP-β-CD inclusion complex was significantly higher than that of Am80. An improved dissolution with approximately 97% drug release in 3 min was observed, in comparison with Am80 with approximately 60% release in 45 min. In vivo studies indicated that the AUC0-∞ has increased 2.79 times and the Cmax 4.37 times after the formation of inclusion complex. Further, the decrease of Cl/F and tmax indicated the Am80-HP-β-CD inclusion complex can be absorbed into blood faster as well as being cleaned out slower. In short, the bio-availability of Am80 was notably increased by complexing it with HP-β-CD.