Our study investigated the differentiation of Th1 and Tfh cells following acute LCMV infection using the adoptive transfer and analysis of LCMV-specific CD4 T cells (SMARTA TCR transgenic) induced by acute viral infection (20). Following the clearance of acute LCMV infection and the corresponding contraction of virus-specific CD4 T cells, both Th1 and Tfh memory subsets are maintained at relatively stable numbers for approximately 60–150 days post-infection (20). Resting PD-1− and ICOS−CXCR5+Ly6clo memory CD4 T cells shared phenotypic and gene expression similarities to CXCR5+Ly6clo effector Tfh cells, suggesting a direct lineage-relationship between these populations at the different stages of the immune response. Adoptive transfer of CXCR5+ memory subsets followed by LCMV Armstrong challenge resulted in Tfh secondary effector cells with ability to rapidly recall a Tfh effector phenotype and promote the generation of germinal center B cells. In addition, when CXCR5+ memory cells were transferred to B cell deficient recipient mice that were then infected, a large proportion of the resulting effector cells recalled and sustained a Tfh-like phenotype, while primary effector cells generated from naïve (uncommitted) SMARTA cells did not. These latter results indicate that CXCR5+ memory cells have acquired and maintained a Tfh-biased cell program relative to their naïve cell counterparts (20). Interestingly, while there was some apparent flexibility by some CXCR5+ memory cells to generate CXCR5− Th1-like secondary effector cells, cell-intrinsic restrictions impaired the Th1 effector program, resulting in poor granzyme B and IFNγ expression. The results from this study argue in favor of Tfh lineage commitment within the CXCR5+ Tfh memory cell population (20).

In contrast to these many studies that report the existence of memory Tfh cells that promptly and preferentially recall Tfh phenotype and function upon rechallenge, one study reported that *L. monocytogenes* specific (identified by MHC class II tetramer) CXCR5+ memory CD4 T cells are pluripotent, promoting the recall of both Tfh cells and non-Tfh (Th1) cells (31). Another study of influenza infection in IL-21 GFP reporter mice showed that the adoptive transfer of polyclonal CXCR5+GFP+ Tfh CD4 effector cells gave rise to memory cells with sufficient plasticity to generate secondary effectors of both Tfh cells and non-Tfh cells. Thus, Luthje et al. conclude that Tfh effector cells are "uncommitted" regarding their T helper lineage (43). Interestingly, these studies both used adoptive transfer of polyclonal CD4 T cells (31, 43), while studies demonstrating relative Tfh commitment of CXCR5+ Tfh memory cells utilized TCR transgenic cells specific for a single epitope (20, 40, 42). Because strength and/or longevity of TCR signaling influences Th1 versus Tfh differentiation (44, 45) as well as memory CD4 T cell differentiation (46), individual TCRs within the Tfh effector population may confer varying degrees of lineage commitment in CXCR5+ Tfh memory cells. Going forward, it will be interesting to determine whether different TCRs, infection and immunization systems, and other factors play a role in the degree of plasticity versus commitment in memory Tfh cells.

There are many well described Tfh cell markers; however, there are relatively few markers useful for clearly distinguishing resting memory Tfh cells. Antigen-specific memory Tfh cells express CXCR5, but lack Bcl6, ICOS, and expression of many other Tfh molecules (20, 41, 47) (**Figure 2**). In addition, memory Tfh cells do not express CD69 (20), an indicator of ongoing antigen stimulation. A recent study demonstrated that folate receptor 4 (FR4), a surface receptor that is upregulated on Tfh cells but not Th1 cells during acute viral infection, is maintained on the surface of CXCR5+ memory Tfh cells (48). To date, the best marker for identifying memory Tfh cells is CXCR5 (18, 20, 41, 47) (**Figure 2**); however, not all CXCR5+ memory cell subsets exhibit specific Tfh function upon reactivation (47). **Figure 2** shows some of the phenotypic markers associated with the memory Tfh cell phenotype.

It has become clear that Tfh effector cells can become memory Tfh cells with cell-intrinsic programs that promote the recall of Tfh cells upon reactivation. It is therefore possible to propose models of T helper cell differentiation wherein naïve CD4 T cells give rise to Tfh and other T effector lineage cells (Th1,Th2,or Th17 depending upon context of the pathogen or inflammation), and these effector cells give rise to memory cells that strictly maintain their lineage commitment and recall their lineage upon reinfection or boosting with antigen (**Figure 3A**). However, memory T cell populations are characterized by a vast degree of phenotypic and functional heterogeneity. A second and more comprehensive model would suggest that as effector cells differentiate, individual cells acquire varying degrees of programing toward the Tfh or other T effector lineage cells that can be stably maintained in resting memory cells (likely through epigenetic mechanisms) (**Figure 3B**). The resulting population of antigen-specific memory cells would then contain a wide spectrum of cells with varying degrees of lineage commitment versus pluripotency/plasticity (**Figure 3B**). This latter model may account for why some studies report lineage commitment within Tfh cell subsets, while others studies report relative noncommitment or plasticity. Future mechanistic studies will provide improved insight into the heterogeneity of memory CD4 T cells in relation to their T helper lineage commitment and recall potential. Furthermore, determining whether Tfh memory cells are homeostatically maintained over time at very late memory timepoints remains to be explored.

#### **HUMAN T FOLLICULAR HELPER MEMORY CELLS**

The original reports describing CXCR5+ Tfh cells in human tonsils had differing ideas regarding the existence of memory Tfh cells. One report described that tetanus-antigen-specific proliferation was induced in CD4+CD45RO+CXCR5− but not CD4+CD45RO+CXCR5+ cells, thus suggesting that Tfh cells likely disappear along with germinal centers, and are prone to apoptosis due to their high levels of Fas expression (4). In contrast, the other report showed that unlike the activated tonsillar CXCR5+ CD4 T cells that were CD69+HLA-DR+ICOS+, circulating CXCR5+ cells in human blood are CD69−HLA-DR− ICOS−, and hypothesized that these blood CXCR5+ cells represent memory Tfh cells (5). Since these initial observations, whether activated human Tfh cells become memory T cells, and whether

activation dependent molecules. Bcl6 expression is not detected in memory Tfh cells; however, Maf and other transcription factors are maintained at low/intermediate levels. PD-1 is absent from antigen-specific memory Tfh cells in mice, however, human CXCR5+CXCR3− memory Tfh cells maintain low levels of PD-1 expression that is not dependent on TCR signaling. Folate receptor 4 (FR4), a molecule that is highly expressed on effector Tfh cells, is maintained on CXCR5+ memory Tfh cells.

they maintain their Tfh function upon reactivation has remained unclear. Recent reports have shed light on the nature of human CXCR5+ memory Tfh cell ontogeny and function (18, 37, 47).

One study described a population of circulating human CXCR5+ central memory CD4 T cells that expressed CXCL13 and promoted B cells to undergo plasma cell differentiation and Ig secretion (37). Another recent study investigating circulating CXCR5+ CD4 T cells in human blood revealed that these cells share functional characteristics with Tfh cells. CXCR5+ cells promoted the isotype switching and antibody production of IgG, IgA, and IgE isotype switched antibodies in T:B cell co-culture experiments, while CXCR5− cells did not promote switched antibody production (18). Their study further categorized the CXCR5+ population into CXCR3+CCR6− (Th1-like), CXCR3−CCR6+ (TH17-like), and CXCR3−CCR6− (Th2-like) subsets. While CXCR5+CXCR3+CCR6− cells did not provide help in these co-culture assays, CXCR5+CXCR3−CCR6+ cells promoted high levels of IgG and IgA antibodies, while CXCR5+CXCR3−CCR6− cells promoted high IgG and IgE, and intermediate levels of IgA. This study highlights the function of reactivated CXCR5+ CD4 T cells from peripheral blood to promote antibody production, and further defines the vast heterogeneity of CXCR5+ cells in relation to the Th1, Th2, and Th17 lineage of cells (18). The CD69− and ICOS− phenotype of these circulating CXCR5+ cells (prior to reactivation) suggests that they are resting memory cells (18). However, inability to identify antigen-specific cells and relate them to a known time of antigen exposure lead to difficulty in concluding whether they are indeed true memory Tfh cells that persist long-term in the absence of antigen and maintain their lineage characteristics in their resting state.

Locci et al. profoundly advanced the understanding of human Tfh memory cells and clarified their existence. Their study identified that blood CXCR5+CXCR3− cells that had a nonactivated/resting phenotype could be stratified into either PD-1− or PD-1low subsets (47). Interestingly, low PD-1 expression was stably maintained on this latter subset of cells during 20 day culture *in vitro* in the absence of TCR stimulation, suggesting that sorted CXCR5+PD-1low cells express these molecules (PD-1 and CXCR5) as a part of the Tfh transcriptional program. Indeed, the transcriptional profile of these CXCR5+CXCR3−PD-1low cells is similar (albeit reduced) to that of germinal center Tfh cells from human tonsils, and these cells promoted the highest IgG antibody production and plasmablast differentiation in T:B cell co-culture experiments compared to other subsets of CXCR5+ cells (47). Furthermore, characterization of tetanus-specific CD4 T cells in healthy human donors (identified using HLA tetramers bearing tetanus peptide), demonstrated the existence of this CXCR5+CXCR3−PD-1low phenotype among resting antigenspecific memory cells (**Figure 2**). Together, these experiments define the phenotype and function of human memory Tfh cells and combine to fortify the idea that memory Tfh cells exist within the pool of resting human antigen-specific memory cells (47).

Defining strategies and markers to distinguish and characterize antigen-specific human Tfh effector and memory subsets is difficult, and is the focus of many recent and current lines of research (18, 47, 49–51). The vast heterogeneity of effector and memory CD4 T cells in human blood and other tissues has led to difficulty in understanding what Tfh-like cell subsets serve as the best correlates of effective antibody responses, and what subsets actually provide the help for the B cell response *in vivo* (51). Some of the differences for Tfh and/or Tfh memory markers utilized in these various studies may result from the different types of infections or vaccinations studied, the timepoints analyzed (early after vaccination versus long-lived memory cells), and the varying analysis of "bulk" memory populations of unknown antigen-specificities versus analysis of tetramer+ pathogen specific memory cells (18, 47, 49–51). As better markers, reagents, and methods for studying antigen-specific Tfh responses become available, the developmental requirements and heterogeneity of human memory Tfh cells and their relationship to other T helper cell subsets will be better understood. This will open doors to more clearly define the specific roles that Tfh memory cells play during recall responses by human Tfh cells during boosting or in response to natural infection.

#### **PRESERVING THE Tfh PROGRAM IN RESTING MEMORY Tfh CELLS**

A complex combination of cytokines, transcription factors (52), STAT molecules (53), and epigenetic changes (54, 55) combine to delineate both the initial differentiation and stability of T helper lineages. Extrinsic factors that drive CD4 T cell activation and T helper cell differentiation are absent after antigen/pathogen clearance. Consequently, transcription factors such as Tbet and Bcl6 and other molecules that define T helper lineages are downregulated on memory CD4 T cells (20, 31, 40, 41). A critical question regarding the existence of CXCR5+ Tfh memory cells (and other specialized T helper cell memory subsets) remains: how do memory cells maintain lineage commitment and remember the gene expression programs of their previously defined T helper lineage in the absence of antigen and inflammatory signals (**Figure 4A**) (56, 57)? While the answer to this question for memory Tfh cells is currently not resolved, it is likely that the

Tfh secondary effectors cells upon reactivation with antigen. In contrast, uncommitted naïve CD4 T cells are pluripotent, with the capacity to generate a balance of Tfh and non-Tfh (Th1, Th2, Th17) effector cells following activation. **(B)** The maintenance of Tfh associated transcription factors may promote the recall of the Tfh cell program and inhibit the differentiation of other T helper lineages following activation. The transcription factor Maf, is maintained at low levels in human Tfh memory cells. While Bcl6 expression is not detectable in memory Tfh cells, it is possible that soon after reactivation, rapid reexpression of Bcl6 directs Tfh gene expression as well as inhibits Blimp1 and high expression of Tbet,

with B cells that can reinforce the Tfh cell phenotype and function. In contrast, CCR7+ (CXCR5−) naïve CD4 T cells will become activated in T cells zones by antigen-bearing dendritic cells. **(D)** Epigenetic programs acquired during Tfh effector differentiation can be maintained in resting memory Tfh cells throughout homeostatic proliferation. Transcriptionally permissive histone modifications and unmethylated DNA at "poised" Tfh associated loci in memory Tfh cells will promote the rapid reexpression of these genes upon reactivation. In addition, repressive histone modifications and DNA methylation will prevent reexpression of Th1, Th2, and Th17-associated genes.

reexpression of key transcription factors, as well as the epigenetic state of genes related to Tfh cell development and function, play an important role in directing Tfh memory cells toward recalling a secondary Tfh response. Potential mechanisms (that are not mutually exclusive – and more likely work in concert) for preserving the program of Tfh memory cells that promote the recall of Tfh secondary effector cells are shown in **Figures 4B–D**.

Bcl6 plays a critical role in defining the phenotype and function of Tfh cells (8–10). Although all of the mechanisms by which this transcriptional repressor positively directs the Tfh gene expression program, Bcl6 expression can induce CXCR5 expression (9, 15), likely through an indirect mechanism. Bcl6 overexpression in naïve human CD4 T cells promotes the regulation of genes required for germinal center trafficking and interactions with B cells, and in cooperation with Maf induces expression of other key Tfh genes (15). It is clear that while many Tfh associated genes are downregulated in resting CXCR5+ memory cells, these cells maintain a Tfh-like gene expression profile for many other Tfh associated genes (20, 47). Bcl6 transcript was not detectable in memory Tfh cells (20, 41, 42, 47). However, this does not rule out that very low levels of Bcl6 expression could bias the recall of memory Tfh cells early after cell reactivation by promoting the Tfh phenotype and inhibiting Blimp1 activity (**Figure 4B**). Furthermore, because memory B cells can present antigen to and rapidly induce Bcl6 expression in memory Tfh cells (58), the Bcl6-dependent Tfh transcriptional program may be robustly reinforced in memory Tfh cells upon reactivation *in vivo*.

Maf gene and protein expression were found at low levels in human blood Tfh memory cells (CXCR5+CXCR3− PD-1+) (47), suggesting that this important transcriptional regulator of Tfh cell functions (14, 15) may play a role in preserving aspects of the Tfh phenotype and program in these memory cells. In addition, Ascl2, a recently described transcription factor was shown to be required for early Tfh differentiation and function (16). Overexpression of Ascl2 results in CXCR5 expression, CCR7 downregulation, and subsequent migration to the follicles. Ascl2 binds to conserved non-coding sequence regions of the *Cxcr5* locus and promotes *Cxcr5* gene expression. Interestingly, Ascl2 did not induce Bcl6 expression. It is therefore possible that Ascl2 expression in Tfh memory cells may be important for maintenance of CXCR5 expression in resting memory Tfh cells (16). Currently, it is unclear what specific roles Bcl6, Maf, Ascl2, or other transcription factors play in preserving CXCR5 expression on memory Tfh cells, and promoting maintenance of the Tfh gene expression program (**Figure 4B**). Upon reactivation, it is possible that CXCR5+ Tfh memory cells are already localized (or rapidly relocalize) to the T:B border, resulting in interactions with B cells that would then reinforce Bcl6 reexpression and other Tfh transcription factors (**Figure 4C**). In turn, this might preferentially promote the Tfh program (over other T helper lineage programs) during the secondary response (**Figure 4C**). Currently, it remains unclear how these and other transcription factors influence the maintenance of the Tfh program and the repression of other T helper cell lineages in Tfh memory cells.

Transcription factors that have been termed lineage "master regulators," such as Tbet, RORγt, and Gata3 are not always limited in their expression to a single subset of differentiated CD4 T cells and can be expressed in Tfh effector cells (**Figure 1**) (1). The expression of these factors in Tfh effector cells is believed to be important for promoting expression of key cytokines that direct specific isotype class switching in cognate germinal center B cells (17, 21, 22). The balance of Bcl6 and Tbet has been shown to have important effects of differentiation and gene expression by Th1 and Tfh cells (59–61). Interestingly, following LCMV infection, mouse Tfh cells co-express Bcl6 and intermediate levels of Tbet (20). The resulting Tfh memory cells do not have detectable Bcl6, but surprisingly, maintain low levels of Tbet gene and protein expression (20). Despite this Tbet expression (and lack of Bcl6), adoptively transferred Tfh memory cells predominantly generate Tfh secondary effector cells following reinfection (20). This finding invokes the idea that beyond the simple balance of lineage-associated transcription factors, transcriptional programing through epigenetic modifications likely plays a role in promoting the reexpression of Tfh genes and repressing the gene expression programs of other T helper cell lineages (57).

The maintenance of gene expression and lineage-differentiation programs in the absence of inflammatory/differentiating signals is reinforced by epigenetic programs that are acquired during the initial effector T cell differentiation (54, 57). The combination of epigenetic modifications to histones to regulate the structure of chromatin and the methylation of DNA at CpG motifs that determine the binding of inhibitory methyl-binding domain proteins can be conserved in daughter cells throughout cell division (62), providing a mechanism for gene expression programs to be maintained during T cell homeostatic proliferation (57). Furthermore, transcriptionally permissive epigenetic marks are positively associated at loci of lineage-specific cytokines for each T helper lineage, while repressive marks at specific loci relevant to other T helper lineages serve to prevent inappropriate gene expression and suppress the differentiation toward alternative lineages (54, 57). Lu et al. observed that positive chromatin marks were associated in Tfh-like cells at the *Tbx21*, *Gata3*, and *Rorc* loci, and that these cells could be repolarized toward Th1, Th2, and Th17 cells when restimulated under polarizing conditions (63). This study highlights that certain features of the Tfh program may partially overlap with Th1, Th2, and Th17 cell programs, possibly allowing greater plasticity toward the capacity to produce multiple lineagedefining cytokines. In addition, *in vitro* differentiated Th1 and Th2 cells (but not Th17 cells) can become Tfh cells following adoptive transfer and immunization with protein in adjuvant (42). Together, these studies demonstrate that there is a degree of flexibility between effector Tfh cells and other T helper cells. Further work is required to determine whether and how such flexibility applies to CXCR5+ memory Tfh cells generated *in vivo*. It is possible that context-dependent selective processes during memory T cell development promote antigen-specific memory T cells that are either more or less committed to Tfh differentiation compared to their effector (precursor) counterparts (**Figure 3**).

Methylation of CpG motifs in regulatory regions of genomic DNA serve as a transcriptionally repressive mark through recruitment of methyl-binding domain proteins (62, 64). Loss of DNA methyltransferase activity has dramatic effects upon CD4 differentiation, resulting in loss of restriction of key lineage-associated cytokine and transcription factor genes (65–69). DNA bisulfite sequencing analysis of LCMV-induced CD4 T cells revealed that the *Gzmb* locus (encoding granzyme B) became demethylated in Th1 but not Tfh effector cells. Furthermore, the maintenance of DNA methylation at the *Gzmb* locus in CXCR5+ memory Tfh cells was predictive of both their capacity to recall the Tfh effector phenotype upon reactivation with antigen *in vivo*, and also their inability to efficiently express granzyme B, even among the small fraction of Th1-like secondary effector cells that had downregulated CXCR5 expression (20). Thus, repression of Th1 associated gene expression programs (such as *Gzmb*) in memory Tfh cells likely plays an important role in the maintenance of the Tfh program in resting CXCR5+ Tfh memory cells by repressing the expression of genes associated with Th1 effector function (**Figure 4D**). Future work is needed to characterize permissive versus repressive epigenetic marks (for loci of lineageassociated cytokines, effector molecules, and transcription factors) in antigen-specific memory Tfh, Th1, Th2, and Th17 cells, providing needed insight into the plasticity versus commitment of memory CD4 T cell subsets induced by infection and immunization.

#### **CONCLUDING REMARKS**

The discovery that phenotypically unique subsets of CXCR5+ memory CD4 T cells have recall potential specific for Tfh function invites important questions for future study that will inform vaccination strategies for infectious diseases. Understanding the mechanisms of how Tfh memory cells acquire and preserve the Tfh gene expression programs and preferentially recall these programs upon reactivation will provide important insight into the lineage maintenance and plasticity of these cells. Finally, future work is needed to determine the optimal ways to utilize Tfh memory cells during prime and boost immunization to promote ways that improve protective and long-lived antibody responses.

#### **ACKNOWLEDGMENTS**

We thank Dr. Ali H. Ellebedy and Dr. Ben Youngblood for helpful discussion. This work was supported by the NIH grant RO1 AI030048 (to Rafi Ahmed) and the National Institute of Allergy and Infectious Diseases grant F32 AI096709 (to J. Scott Hale).

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 17 October 2014; accepted: 08 January 2015; published online: 02 February 2015.*

*Citation: Hale JS and Ahmed R (2015) Memory T follicular helper CD4 T cells. Front. Immunol. 6:16. doi: 10.3389/fimmu.2015.00016*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2015 Hale and Ahmed. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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One of the most promising possibilities for enhancing enzyme activity during immobilization, especially for entrapment methods, is molecular imprinting which conserves by the aid of added substrates or substrate analogues the proper shape of the enzyme's active site [7, 8]. This so-called bioimprinting, as can be rationalized by the generally accepted hypothesis of the interfacial activation mechanism, can influence the biocatalytic activity and enantioselectivity of lipases. The positive effect of bioimprinting on lipases was already demonstrated by immobilizations using sol-gel entrapment [9, 10]. The active site of many lipases in aqueous solution is covered by a flexible region of the enzyme, often referred to as a lid. Interaction of the lid with hydrophobic molecules can enforce its opening to make the active site accessible [11, 12]. This hypothesis is supported by crystal structures of lipases in their open and closed forms [13, 14]. Because interfacial activation can increase significantly the catalytic activity and selectivity of lipases, it should be considered for all applications of lipases including development of novel immobilization methods. Although bioimprinting proved to be an efficient tool to modulate the properties of lipases entrapped in sol-gel matrices, this strategy has been extended for entrapment in electrospun nanofibers only recently in one of our previous studies [7].

To demonstrate the efficiency and generality of bioimprinting during entrapment of lipases, lipase AK and CaLB were entrapped in PVA and polyaspartamide nanofibers in the presence of four different additives as bioimprinting agents. Poly(ethylene glycol)s (PEGs) of different molecular weights and nonionic detergents such as Tween 80 and Brij 30 were already applied as substrate analogues exhibiting bioimprinting effects in sol-gel systems [11] and in PVA nanofibers [6], but their effect in polyaspartamide entrapment has not been investigated yet. To reveal the effect of additives and the difference between fiber-forming polymer matrices, CaLB was chosen as model enzyme for the bioimprinting experiments. Results of testing the immobilized nanofibrous CaLB biocatalysts by kinetic resolution of *rac*-**1** showed that all tested additives increased the specific enzyme activity (*U*E) and the enantiomeric excess of the product (*ee*P) (**Table 2**). The

**Table 2.** Kinetic resolution of 1-phenylethanol (rac-**1**) with CaLB-PVA or CaLB-polyaspartamide nanofibers fabricated in the presence of different additives.

**3. Entrapment of drugs in electrospun nanofibers**

**applications**

**3.1. Encapsulation of rifampicin in polyaspartamide nanofibers for ophthalmic**

The interest in the development of novel ophthalmic drug formulations has increased considerably because of the low bioavailability of drug molecules after their administration on the surface of the eye. The low therapeutic efficiency is caused by the complex structure of the eye, the small absorptive surface, and low transparency of the cornea, lipophilicity of corneal epithelium, metabolism, bonding of the drug to proteins in tear liquid, and protective mechanisms such as tear formation, blinking, and the flow of the active pharmaceutical ingredients (APIs) through the nasolacrimal duct [16, 17]. The main challenge in the development of ophthalmic drug formulations is to achieve the required drug concentration at the site of absorption and to improve residence time, which in turn contributes to smaller application frequency [18, 19].

Electrospun Nanofibers for Entrapment of Biomolecules http://dx.doi.org/10.5772/intechopen.76068 143

Polymers are often used in ophthalmic formulations to increase viscosity and thus, residence time of the formulation on the corneal surface, which increases the bioavailability of the API. Drug penetration can also be enhanced by various additives such as chelating agents, surfactants, and cyclodextrins, which form inclusion complexes leading to increased solubility, permeability, and bioavailability of the poorly soluble drugs. Controlled release of the drug can also be achieved by novel formulations such as inserts, collagen shields, contact lenses, and in situ gels. These solid or semi-solid formulations provide controlled release and increasing bioavailability of the API due to the extended contact time with cornea. Electrospun

nanofibers offer controllable release rate as well as exact and safe dosage of the API.

development of a stable solid formulation of rifampicin is still required.

In our work, a novel solid nanofibrous formulation was designed to encapsulate rifampicin (RFP), an important antibiotic and antiviral agent. Rifampicin inhibits bacterial DNAdependent RNA synthesis; thus, it can be applied for the treatment of many infectious diseases such as tuberculosis and Hansen's disease [20]. Although rifampicin has great efficiency, the scope of its applications is limited by its poor hydrolytic and thermal stability and by its rather limited solubility [21]. Due to these difficulties and the inaccurate dosing of eye drops,

Poly(aspartic acid) (PASP) is a biocompatible and biodegradable synthetic poly(amino acid) having great potential in biomedical applications. For using as a polymer in electrospinning, PASP derivatives, including cationic polyaspartamides, can be synthesized under mild reaction conditions [22, 23]. The biological activity of the PASP-based polymers has been investigated carefully and, owing to their protein-like structure, they are expected to be biodegradable. Due to these advantageous properties of polyaspartamide derivatives, rifampicin can be beneficially entrapped in polyaspartamide-based nanofibers fabricated by electrospinning. Poly(aspartic acid) derivatives with butyl and hexyl groups were tested to determine the effect of chemical structure on release properties. For electrospinning of PASP derivatives containing rifampicin (1.6 w/w%), ethanol was used as the solvent to avoid the degradation of the active compound. The morphology of electrospun fibers was investigated by SEM indicating that uniform nanofibers could be produced independently of chemical composition. By using butyl side groups, the fiber diameter was ~800 nm, while

**Figure 7.** Dynamic viscosity and glass transition temperature (*T*g ) of electrospun nanofibers (a) from PVA- and (b) from polyaspartamide-based precursor systems.

best results were obtained with PEG 400 and Tween 80 as additives in electrospinning of CaLBpolyaspartamide fibers, while, in case of electrospinning of CaLB-polyaspartamide fibers, PEG 400 and PEG 1000 were the most efficient bioimprinting agents. It must be noted, that CaLB in polyaspartamide polymer matrix without additive exhibited much higher activity than in PVA. Consequently, additives could manifest their beneficial effect on the activity of CaLB in polyaspartamide nanofibers better than in PVA nanofibers.

The polymer matrix of nanofibers is able to influence the enzyme activity by the bioimprinting effect. In addition, the physicochemical properties of the matrix material may affect also significantly the apparent enzyme activity and the final properties of immobilized biocatalyst. Interactions between polymer chains can affect significantly the diffusion limitations for substrate or product, which can strongly influence the apparent efficiency of the immobilized biocatalyst. Thus, dynamic viscosity as a rheological property of the precursor systems (enzyme-polymer-additive mixtures) and glass transition temperature (*T*g ) as a thermal property of the resulted enzyme-filled nanofibrous materials were also investigated (**Figure 7**). According to Kramers' theory, the biocatalytic activity of enzymes could strongly depend on the viscosity of solvent because viscosity results in friction of proteins in solution leading to decreased motion and inhibiting catalysis by motile enzymes [15]. Thus, viscosity of the precursor system could affect directly the properties of the immobilized enzyme during its entrapment. Moreover, the viscosity of the polymer precursor system can significantly influence fiber formation during electrospinning as well. The dynamic viscosity values of PVA- and polyaspartamide-based precursor systems were determined in the presence of CaLB and the best additives such as PEG 400, PEG 1000, and Tween 80 and were compared to the simple polymer solution without the enzyme or additive. The glass transition temperature of the electrospun products was investigated as well. In case of CaLB-PVA systems, PEG 400 slightly increased the viscosity but Tween 80 resulted in higher increase in the viscosity value. On the other hand, when *T*g values of the different CaLB-PVA fibrous biocatalysts were compared, PEG 400 had more pronounced effects on the interaction between the polymer chains, while the *T*g of PEG 400-containing CaLB-PVA fibers was significantly lower than that of the CaLB-PVA fibers without additives (**Figure 7a**). In case of CaLB-polyaspartamide, additives (PEG 400 and PEG 1000) had no significant effect on the viscosity of precursor system, but *T*g values became lower due to the presence of PEG molecules, especially in the case of PEG 1000 (**Figure 7b**).

## **3. Entrapment of drugs in electrospun nanofibers**

best results were obtained with PEG 400 and Tween 80 as additives in electrospinning of CaLBpolyaspartamide fibers, while, in case of electrospinning of CaLB-polyaspartamide fibers, PEG 400 and PEG 1000 were the most efficient bioimprinting agents. It must be noted, that CaLB in polyaspartamide polymer matrix without additive exhibited much higher activity than in PVA. Consequently, additives could manifest their beneficial effect on the activity of CaLB in

The polymer matrix of nanofibers is able to influence the enzyme activity by the bioimprinting effect. In addition, the physicochemical properties of the matrix material may affect also significantly the apparent enzyme activity and the final properties of immobilized biocatalyst. Interactions between polymer chains can affect significantly the diffusion limitations for substrate or product, which can strongly influence the apparent efficiency of the immobilized biocatalyst. Thus, dynamic viscosity as a rheological property of the precursor sys-

property of the resulted enzyme-filled nanofibrous materials were also investigated (**Figure 7**). According to Kramers' theory, the biocatalytic activity of enzymes could strongly depend on the viscosity of solvent because viscosity results in friction of proteins in solution leading to decreased motion and inhibiting catalysis by motile enzymes [15]. Thus, viscosity of the precursor system could affect directly the properties of the immobilized enzyme during its entrapment. Moreover, the viscosity of the polymer precursor system can significantly influence fiber formation during electrospinning as well. The dynamic viscosity values of PVA- and polyaspartamide-based precursor systems were determined in the presence of CaLB and the best additives such as PEG 400, PEG 1000, and Tween 80 and were compared to the simple polymer solution without the enzyme or additive. The glass transition temperature of the electrospun products was investigated as well. In case of CaLB-PVA systems, PEG 400 slightly increased the viscosity but Tween 80 resulted in higher increase in the viscosity value. On the other

values of the different CaLB-PVA fibrous biocatalysts were compared, PEG 400

had more pronounced effects on the interaction between the polymer chains, while the *T*g

due to the presence of PEG molecules, especially in the case of PEG 1000 (**Figure 7b**).

1000) had no significant effect on the viscosity of precursor system, but *T*g

PEG 400-containing CaLB-PVA fibers was significantly lower than that of the CaLB-PVA fibers without additives (**Figure 7a**). In case of CaLB-polyaspartamide, additives (PEG 400 and PEG

) as a thermal

) of electrospun nanofibers (a) from PVA- and (b) from

of

values became lower

tems (enzyme-polymer-additive mixtures) and glass transition temperature (*T*g

polyaspartamide nanofibers better than in PVA nanofibers.

**Figure 7.** Dynamic viscosity and glass transition temperature (*T*g

142 Electrospinning Method Used to Create Functional Nanocomposites Films

polyaspartamide-based precursor systems.

hand, when *T*g

#### **3.1. Encapsulation of rifampicin in polyaspartamide nanofibers for ophthalmic applications**

The interest in the development of novel ophthalmic drug formulations has increased considerably because of the low bioavailability of drug molecules after their administration on the surface of the eye. The low therapeutic efficiency is caused by the complex structure of the eye, the small absorptive surface, and low transparency of the cornea, lipophilicity of corneal epithelium, metabolism, bonding of the drug to proteins in tear liquid, and protective mechanisms such as tear formation, blinking, and the flow of the active pharmaceutical ingredients (APIs) through the nasolacrimal duct [16, 17]. The main challenge in the development of ophthalmic drug formulations is to achieve the required drug concentration at the site of absorption and to improve residence time, which in turn contributes to smaller application frequency [18, 19].

Polymers are often used in ophthalmic formulations to increase viscosity and thus, residence time of the formulation on the corneal surface, which increases the bioavailability of the API. Drug penetration can also be enhanced by various additives such as chelating agents, surfactants, and cyclodextrins, which form inclusion complexes leading to increased solubility, permeability, and bioavailability of the poorly soluble drugs. Controlled release of the drug can also be achieved by novel formulations such as inserts, collagen shields, contact lenses, and in situ gels. These solid or semi-solid formulations provide controlled release and increasing bioavailability of the API due to the extended contact time with cornea. Electrospun nanofibers offer controllable release rate as well as exact and safe dosage of the API.

In our work, a novel solid nanofibrous formulation was designed to encapsulate rifampicin (RFP), an important antibiotic and antiviral agent. Rifampicin inhibits bacterial DNAdependent RNA synthesis; thus, it can be applied for the treatment of many infectious diseases such as tuberculosis and Hansen's disease [20]. Although rifampicin has great efficiency, the scope of its applications is limited by its poor hydrolytic and thermal stability and by its rather limited solubility [21]. Due to these difficulties and the inaccurate dosing of eye drops, development of a stable solid formulation of rifampicin is still required.

Poly(aspartic acid) (PASP) is a biocompatible and biodegradable synthetic poly(amino acid) having great potential in biomedical applications. For using as a polymer in electrospinning, PASP derivatives, including cationic polyaspartamides, can be synthesized under mild reaction conditions [22, 23]. The biological activity of the PASP-based polymers has been investigated carefully and, owing to their protein-like structure, they are expected to be biodegradable. Due to these advantageous properties of polyaspartamide derivatives, rifampicin can be beneficially entrapped in polyaspartamide-based nanofibers fabricated by electrospinning. Poly(aspartic acid) derivatives with butyl and hexyl groups were tested to determine the effect of chemical structure on release properties. For electrospinning of PASP derivatives containing rifampicin (1.6 w/w%), ethanol was used as the solvent to avoid the degradation of the active compound. The morphology of electrospun fibers was investigated by SEM indicating that uniform nanofibers could be produced independently of chemical composition. By using butyl side groups, the fiber diameter was ~800 nm, while

**Acknowledgements**

**Conflict of interest**

**Author details**

András Szilágyi2

Budapest, Hungary

**References**

accounts.7b00218

The authors declare no conflict of interest.

Diána Balogh-Weiser1,2,3, Csaba Németh2

and Economics, Budapest, Hungary

3 SynBiocat Ltd., Budapest, Hungary

5 Spinsplit Ltd., Budapest, Hungary

and László Poppe1,3,6\*

\*Address all correspondence to<EMAIL_ADDRESS>
Technology and Economics, Budapest, Hungary

This research was supported by the National Research, Development, and Innovation Office— NKFIH (FK 125074). Benjámin Gyarmati acknowledges the János Bolyai Research Scholarship of the Hungarian Academy of Sciences for financial support. A. Szilágyi is grateful to the support of the ÚNKP-17-4-III New National Excellence Program of the Ministry of Human Capacities. We thank Anna Szabó for her cooperation in the electrospinning of polyaspartamides (Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, BME) and Imre Miklós Szilágyi (Department of Inorganic and

1 Department of Organic Chemistry and Technology, Budapest University of Technology

2 Department of Physical Chemistry and Materials Science, Budapest University of

4 Department of Electron Devices, Budapest University of Technology and Economics,

6 Faculty of Chemistry and Chemical Engineering, Biocatalysis and Biotransformation Research Centre, Babeș-Bolyai University of Cluj-Napoca, Cluj-Napoca, Romania

[1] Xue J, Xie J, Liu W, Xia Y. Electrospun nanofibers: New concepts, materials, and applications. Accounts in Chemical Research. 2017;**50**:1976-1987. DOI: 10.1021/acs.

, Ferenc Ender4,5, Benjámin Gyarmati2

,

Electrospun Nanofibers for Entrapment of Biomolecules http://dx.doi.org/10.5772/intechopen.76068 145

Analytical Chemistry, BME) for performing the XRD measurements.

**Figure 8.** (a) Release of rifampicin (RFP) from butyl-PASP and hexyl-PASP electrospun matrices and (b) X-ray diffraction spectra of free rifampicin, butyl-PASP electrospun matrix, and rifampicin entrapped in butyl-PASP.

in the presence of hexyl groups, the average diameter of the fibers was ~900 nm. The results of release experiments (**Figure 8a**) showed that the dissolution rate of rifampicin was larger by using butyl-PASP than hexyl-PASP, but in both cases, the total amount of the encapsulated drug was released.

The crystallinity of entrapped API determines its bioavailability; thus, X-ray diffractograms were recorded for the matrices and the native drug. From the comparison of the X-ray spectra of native and encapsulated rifampicin (butyl-PASP + RFP), it can be clearly seen that the originally crystalline rifampicin became amorphous during electrospinning, which can improve the bioavailability even further (**Figure 8b**).

### **4. Conclusion**

Electrospinning is a promising tool for efficient entrapment of biomolecules, such as expensive enzymes or active pharmaceutical ingredients, within polymer nanofibers which are applicable for biotechnology or biomedical purposes. By rational selection of polymer excipients, both small and macromolecules can be immobilized into electrospun matrices and controllable retention and release can be achieved. Poly(vinyl alcohol) (PVA) and polyaspartamide nanofibers are promising candidates to encapsulate enzymes. In case of industrially relevant lipases, such as lipase from *Candida antarctica* (CaLB) or *Pseudomonas fluorescens* (lipase AK), the optimal enzyme loading of nanofibers generated by electrospinning was 5% entrapped in PVA or polyaspartamide matrices. Entrapment of lipases in nanofibers in the presence of different substrate-like additives led to bioimprinting, which could significantly increase the activity and enantioselectivity of the entrapped enzyme. Polyaspartamides could also be used to encapsulate active pharmaceutical ingredients by electrospinning. The encapsulation of rifampicin proved that the chemical modification of PASP with different alkyl side groups had a significant effect on the release rate of APIs, opening up new possibilities for the improvement of therapeutic efficiency with solid dosage forms.

## **Acknowledgements**

This research was supported by the National Research, Development, and Innovation Office— NKFIH (FK 125074). Benjámin Gyarmati acknowledges the János Bolyai Research Scholarship of the Hungarian Academy of Sciences for financial support. A. Szilágyi is grateful to the support of the ÚNKP-17-4-III New National Excellence Program of the Ministry of Human Capacities. We thank Anna Szabó for her cooperation in the electrospinning of polyaspartamides (Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, BME) and Imre Miklós Szilágyi (Department of Inorganic and Analytical Chemistry, BME) for performing the XRD measurements.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

in the presence of hexyl groups, the average diameter of the fibers was ~900 nm. The results of release experiments (**Figure 8a**) showed that the dissolution rate of rifampicin was larger by using butyl-PASP than hexyl-PASP, but in both cases, the total amount of the encapsu-

**Figure 8.** (a) Release of rifampicin (RFP) from butyl-PASP and hexyl-PASP electrospun matrices and (b) X-ray diffraction

spectra of free rifampicin, butyl-PASP electrospun matrix, and rifampicin entrapped in butyl-PASP.

The crystallinity of entrapped API determines its bioavailability; thus, X-ray diffractograms were recorded for the matrices and the native drug. From the comparison of the X-ray spectra of native and encapsulated rifampicin (butyl-PASP + RFP), it can be clearly seen that the originally crystalline rifampicin became amorphous during electrospinning, which can improve

Electrospinning is a promising tool for efficient entrapment of biomolecules, such as expensive enzymes or active pharmaceutical ingredients, within polymer nanofibers which are applicable for biotechnology or biomedical purposes. By rational selection of polymer excipients, both small and macromolecules can be immobilized into electrospun matrices and controllable retention and release can be achieved. Poly(vinyl alcohol) (PVA) and polyaspartamide nanofibers are promising candidates to encapsulate enzymes. In case of industrially relevant lipases, such as lipase from *Candida antarctica* (CaLB) or *Pseudomonas fluorescens* (lipase AK), the optimal enzyme loading of nanofibers generated by electrospinning was 5% entrapped in PVA or polyaspartamide matrices. Entrapment of lipases in nanofibers in the presence of different substrate-like additives led to bioimprinting, which could significantly increase the activity and enantioselectivity of the entrapped enzyme. Polyaspartamides could also be used to encapsulate active pharmaceutical ingredients by electrospinning. The encapsulation of rifampicin proved that the chemical modification of PASP with different alkyl side groups had a significant effect on the release rate of APIs, opening up new possibilities for the

lated drug was released.

**4. Conclusion**

the bioavailability even further (**Figure 8b**).

144 Electrospinning Method Used to Create Functional Nanocomposites Films

improvement of therapeutic efficiency with solid dosage forms.

Diána Balogh-Weiser1,2,3, Csaba Németh2 , Ferenc Ender4,5, Benjámin Gyarmati2 , András Szilágyi2 and László Poppe1,3,6\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary

2 Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, Budapest, Hungary

3 SynBiocat Ltd., Budapest, Hungary

4 Department of Electron Devices, Budapest University of Technology and Economics, Budapest, Hungary

5 Spinsplit Ltd., Budapest, Hungary

6 Faculty of Chemistry and Chemical Engineering, Biocatalysis and Biotransformation Research Centre, Babeș-Bolyai University of Cluj-Napoca, Cluj-Napoca, Romania

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[17] Pahuja P, Arora S, Pawar P. Ocular drug delivery system: A reference to natural polymers. Expert Opinion on Drug Delivery. 2012;**9**:837-861. DOI: 10.1517/17425247.2012.690733

[18] Karthikeyan D, Bhowmick M, Pandeyetal VP. The concept of ocular inserts as drug delivery systems: An overview. Asian Journal of Pharmaceutics. 2008;**2**:192-200. DOI:

[19] Kumari A, Sharma PK, Garg VK, G. Ocular inserts—Advancement in therapy of eye diseases. Journal of Advanced Pharmaceutical Technology and Research. 2010;**1**:291-296.

[20] Calvori C, Frontali L, Leoni L, Tecce G. Effect of rifamycin on protein synthesis. Nature.

[21] Shishoo CJ, Shah SA, Rathod IS, Savale SS, Kotecha JS, Shah PB. Stability of rifampicin in dissolution medium in presence of isoniazid. International Journal of Pharmaceutics.

[22] Krisch E, Gyarmati B, Szilágyi A. Preparation of pH-responsive poly(aspartic acid) nanogels in inverse emulsion. Periodica Polytechnica-Chemical Engineering. 2017;**61**:19-26.

[23] Németh C, Szabó D, Gyarmati B, Gerasimov AV, Varfolomeev M, László K, Szilágyi A. Effect of side groups on the properties of cationic polyaspartamides. European Polymer

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[2] Doshi J, Reneker DH. Electrospinning process and applications of electrospun fibers.

[3] Yarin AL. Taylor cone and jetting from liquid droplets in electrospinning of nanofibers.

[4] Ramakrishna S, Fujihara K, Teo WE, Lim TC, Ma Z. Electrospinning process. In: An Introduction to Electrospinning and Nanofibers. Singapore: World Scientific Publishing

[5] Sóti P, Weiser D, Vígh T, Nagy Z, Poppe L, Marosi G. Electrospun polylactic acid and polyvinyl alcohol fibers as efficient and stable nanomaterials for immobilization of lipases. Bioprocess and Biosystems Engineering. 2016;**39**:449-459. DOI: 10.1007/

[6] Weiser D, Sóti P, Bánóczi G, Bódai V, Kiss B, Gellért Á, Nagy ZK, Koczka B, Szilágyi A, Marosi G, Poppe L. Bioimprinted lipases in PVA nanofibers as efficient immobilized

[7] Dickey FH. The preparation of specific adsorbent. Proceedings of the National Academy of Sciences of the United States of America. 1949;**35**:227-229. http://www.pnas.org/

[8] Mingarro I, Abad C, Braco L. Interfacial activation-based molecular bioimprinting of lipolytic enzymes. Proceedings of the National Academy of Sciences of the United States

[9] Cao X, Yang J, Shu L, Yu B, Yan Y. Improving esterification activity of *Burkholderia cepacia* lipase encapsulated in silica by bioimprinting with substrate analogues. Process

[10] Hellner G, Boros Z, Tomin A, Poppe L. Novel sol-gel lipases by designed bioimprinting for continuous-flow kinetic resolutions. Advanced Synthesis & Catalysis. 2011;**353**:

[11] Verger R. Interfacial activation' of lipases: Facts and artifacts. Trends in Biotechnology.

[12] Reis P, Holmberg K, Watzke H, Leser ME, Miller R. Lipases at interfaces: A review. Advances in Colloid Interface. Sciences. 2009;**147-148**:237-250. DOI: 10.1016/j.cis.2008.06.001

[13] Brady L, Brzozowski AM, Derewenda ZS, Dodson E, Dodson G, Tolley S, Turkenburg JP, Christiansen L, Hugejensen B, Norskov L, Thim L, Menge U. Nature. 1990;**343**:767-770.

[14] Schrag JD, Li Y, Wu S, Cygler M. Ser-His-Glu triad forms the catalytic site of the lipase from Geotrichum candidum. Nature. 1991;**351**:761-764. DOI: 10.1038/351761a0

[15] Uribe S, Sampedro JG. Measuring solution viscosity and its effect on enzyme activity.

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146 Electrospinning Method Used to Create Functional Nanocomposites Films

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s00449-015-1528-y

content/35/5/227

## *Edited by Tomasz Tański, Pawel Jarka and Wiktor Matysiak*

The most effective method of producing nanofibres is the technology of producing in the electrostatic field, which does not require the use of complicated procedures and equipment. Electrospinning allows to produce 1D nanostructures on an industrial scale in a relatively easy and quick way. The method of electrospinning shares the most features with classical technologies in obtaining synthetic fibres that enable forming and generating a stream of previously dissolved or melted polymer and its coaxial stretching, combined with the transition of the polymer from a liquid state to a solid state. In view of the large application possibilities of electrospun fibres, electrospinning is enjoying a dynamically growing interest of scientists, which can be proven by the increasing trend of scientific publications.

Published in London, UK © 2018 IntechOpen © weisschr / iStock

Electrospinning Method Used to Create Functional Nanocomposites Films

Electrospinning Method

Used to Create Functional

Nanocomposites Films

*Edited by Tomasz Tański,*

*Pawel Jarka and Wiktor Matysiak*


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## **Unmanned Maritime Systems for Search and Rescue**

Aníbal Matos, Eduardo Silva, José Almeida, Alfredo Martins, Hugo Ferreira, Bruno Ferreira, José Alves, André Dias, Stefano Fioravanti, Daniele Bertin and Victor Lobo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69492

#### **Abstract**

The development of maritime unmanned tools for search and rescue operations is not a trivial task. A great part of maritime unmanned systems developed did not target such application, being more focused on environmental monitoring, surveillance or defence. In opposition to these applications, search and rescue operations need to take into account relevant issues such as the presence of people or other vessels on the water. Building upon user requirements and overall integrated components for assisted rescue and unmanned search operations (ICARUS) system architecture, this chapter addresses the development of unmanned maritime systems. It starts with an overview of the approach where a two‐ tier solution was adopted to address safety issues and then proceeds to detail each of the developed technologies.

**Keywords:** unmanned marine systems, unmanned surface vehicles

### **1. Introduction**

During maritime search and rescue operations, the safety of the rescuers is a major issue and must be ensured in any circumstance. Therefore, these teams are often forced to adapt, or even to suspend their operations due to external factors and conditions, such as lack of vis‐ ibility or atmospheric and/or maritime adverse conditions. On the other hand, it should be pointed out that rescue response time is a major factor for success in these operations, due to the reduced survival time of victims that fall overboard.

Robotic assets can therefore complement the role of search and rescue teams, as they can operate in dangerous scenarios and under adverse environmental conditions without putting human

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

lives in danger. There is nowadays a broad range of unmanned maritime systems (UMS) that can operate under different environmental conditions, transport a multitude of payload sensing systems and perform distinctive missions [1]. Concerning maritime robotic tools for search and rescue operations, two works are worth mentioning the emergency integrated lifesaving lan‐ yard (EMILY) system [2] and the autonomous galileo‐supported rescue vessel for persons over‐ board (AGAPAS) project [3]. EMILY (emergency integrated lifesaving lanyard) is a remotely operated autonomous vessel that aims to assist the life guards in crowded beaches, providing them a safe and fast response means. AGAPAS is a project orientated, specifically to person overboard situations, where an automatic system perceives that someone fell from the vessel and deploys an unmanned surface vehicle (USV) capable of fetching that.

While these systems are operated in an independent way, within the ICARUS project, par‐ ticularly in the maritime scenario, multiple heterogeneous unmanned platforms (by air or surface) will co‐operate, in order to detect and assist victims. This chapter addresses the adap‐ tations of general purpose UMS and the development of novel assets, performed within the scope of the ICARUS project, to obtain an integrated system able to respond to search and rescue requirements in complex and challenging environments.

#### **2. Overall concepts of operation and platforms**

The assistance of UMS in search and rescue operations may include providing means for the floatation and thermal protection, preventing from fatigue, drowning or hypothermia, thereby increasing the survival rate. Furthermore, when the conditions do not permit the manned search and rescue operations, a fast and effective operation within the disaster sce‐ nario by the robotic assets makes it possible for the rescuers to evaluate and remotely assist the victims before resuming action as soon as the safety conditions are ensured.

Within the scope of the ICARUS project, a complementary approach for the use of UMS was followed. It consisted in having two classes of UMS, large and fast systems, able to arrive to the disaster area in a short time, and small and slower systems, able to get close to survivors on the water providing them floatation and thermal protection without putting them in danger.

For the larger and fast systems, two different platforms were considered: U‐Ranger and Roaz II. U‐Ranger is a 7 m long UMS, weighting more than 1000 kg and able to reach top speed exceeding 40 kts. Roaz II is a 4 m long, weighting up to 400 kg and able to reach a maximum velocity of 10 kts. These were existing platforms operated by partners of the ICARUS consor‐ tium, respectively, Calzoni and INESC TEC. Within the scope of this project, both these plat‐ forms were adapted for search and rescue operations, by integrating on them adequate sensor suites, by endowing them with autonomous behaviours suited for these operations and by incorporating on them the ability to carry and deploy on site smaller platforms.

The smaller platforms are the unmanned capsules (UCAPs) and were completely developed during the project and consist of 1.5 m long UMS, weighting up to 40 kg. Each of these vessels can be remotely operated or execute autonomous missions and carries on its deck an unin‐ flated life raft. Upon reaching survivors, an automatic inflation of the life raft is performed allowing the survivors to jump it on board.

## **3. U‐Ranger USV**

The U‐Ranger (**Figure 1**) is a remotely controlled unmanned surface vehicle (USV) mainly tailored for harbour and ship protection, able to perform intelligence, surveillance and recon‐ naissance (IRS) operations and patrolling of pre‐defined areas.

The U‐Ranger can be equipped with different kinds of sensors like cameras and radar for surface area control, sonar sensors for underwater control and other sensors for environment control. **Table 1** lists the main technical characteristics of the system.

Within the scope of the ICARUS project, the U‐Ranger USV was equipped with a sensor and autonomous behaviour payload from Centre for Maritime Research and Experimentation (CMRE) [4]. The autonomous behaviour payload is based on the mission oriented operat‐ ing suite (MOOS) open‐source open architecture. The MOOS is a C++ cross platform middle‐ ware for robotics research. Its advantages include open source, flexibility, capacity for system growth, functionality across all platforms, a large user community who contribute MOOS architecture modules for all to share, scalability through distributed computation, protocols exist and need not be developed and considerable use in autonomous systems internationally, at CMRE and elsewhere. MOOS interacts with hardware and operator GUI through MOOS interface drivers and MOOS processes (autonomous behaviour sets), each of which is an inde‐ pendent process linked to a central MOOS database by standard internal process connections. Processes post data to the central MOOS database for access by any other process. Processes

**Figure 1.** Calzoni U‐Ranger USV (source: ICARUS).

**Table 1.** Calzoni U‐Ranger technical specification.

subscribe to the data they require, drawing it from the MOOS database on notification of updates. Communications between the sensor/behaviour payload and the control station on‐ shore rely on a worldwide interoperability for microwave access (WiMAX) link, while the very high frequency (VHF) link bypasses the sensor/behaviour payload allowing direct full manual control of the USV.

The sensor suite includes the following sensors:

The thermal and daylight cameras allow night and day operations. Their fields of view and resolutions are such that it is possible to detect a person in the water at 200 m. While the day‐ light camera is quite sensitive to lightning conditions and in particular to the reflections of sun light on the water surface, the thermal camera can provide useful data almost independently of the environmental conditions. An example of an image provided by this camera can be observed in **Figure 2**. Furthermore, the cameras are mounted on a gyro‐stabilized platform that also allows their pan and tilt command.

The radar installed on the U‐Ranger operates on the X‐Band (9.3–9.4 GHz) and can be config‐ ured with different range settings from 50 m to 24 nautical miles. It has a rotation rate of 24 RPM and it is interfaced to the computational system using public domain C++/Java plugins/ libraries (openCPN BR24 plugin and openbr24 Java). This radar is able to reliably detect obstacles at ranges greater than 50–100 m. The major drawback is the difficulty in detecting fast objects or making detections during sharp turns of the U‐Ranger.

Unmanned Maritime Systems for Search and Rescue http://dx.doi.org/10.5772/intechopen.69492 81

**Figure 2.** Example image from the thermal camera on the U‐Ranger (source: ICARUS).

The laser scanner has four vertically stacked beams with a spacing of 0.8°, which are steered within a 110° angle and with 0.125° horizontal resolution. The maximum scanning frequency is 50 Hz and the obstacle detection range is greater than 100 m. This scanner is mounted near the U‐Ranger bow on a gyro‐stabilized platform (**Figure 3**).

The behaviour set implemented in the U‐Ranger MOOS system contains the following elements:

	- Classic part of the MOOS‐IvP and always active.
	- U‐Ranger vehicle is not able to stop in an autonomous mode.
	- Vehicle with a single thruster and rudder.
	- Last waypoint is always considered as a station keeping point.
	- Selection of waypoints is the C2I operator responsibility.
	- Any waypoint can be selected as the U‐CAP deployment point except the last one..
	- Pre‐defined operational area polygon.

The available behaviours are combined in real time by a function optimizer in order to deter‐ mine the direction the USV should take.

**Figure 3.** Laser scanner mounting platform and electronics (source: ICARUS).

### **4. Roaz II USV**

Roaz II is an USV that can operate in full autonomous mode or remotely operated from a base station. It can be configured to carry different sets of sensors and to perform several kinds of missions, including environmental monitoring, harbour protection or bathymetric data gathering. Its main characteristics are described in **Table 2**.

Roaz II is operated from a mission control station composed by a ruggedized computer and a set of auxiliary devices including antennas. It is capable of executing autonomous missions defined by a list of waypoints differential GPS system and an inertial measurement unit. Telemetry as well as payload data are transmitted in real time to the mission control station.

**Table 2.** Roaz II technical specification.

For navigation purposes, the vehicle uses a precision L1/L2 GPS receiver (Septentrio PolaRx2) and an inertial motion unit (Microstrain 3DM‐GX1) providing attitude informa‐ tion. Propulsion is achieved through the use of two 2 kW electric thrusters with the vehi‐ cle reaching a maximum speed of 10 kts. A set of LiFePo batteries provides up to 8 h of autonomy. Communications with the control station are provided by a WiFi link, allowing remote control, autonomous mission supervision and also transmission of telemetry and payload data to shore.

Although this USV does not fulfil all the requirements for search and rescue (SAR) operations as defined in the ICARUS project (mainly the ones related to maximum speed and range) and its adaptation would require a great effort, its characteristics make it a valuable asset in many experiments as well as in the final demonstration scenario [5].

For that purpose, a thermal camera, a visible camera as well as a radar similar to the one inte‐ grated in the U‐Ranger were also integrated in Roaz II (**Figure 4**).

**Figure 4.** Roaz II USV (source: ICARUS).

#### **5. Unmanned capsule**

The UCAP (**Figure 5**) is a single hull vessel, with a lower rear deck to accommodate the unin‐ flated life raft as well as the corresponding compressed gas bottle. The hull was fabricated in fibreglass, using as custom‐made mould [6].

The UCAP dimension is 1.45 m (length) × 0.52 m (width) × 0.42 m (maximum height). It weighs 22 kg and has a payload capacity exceeding 15 kg.

A jet drive unit assures the propulsion of the UCAP. This jet drive unit is attached to a brush‐ less motor and is capable of delivering a maximum force of 80 N with a power consumption of 800 W. This maximum thrust assures a top speed greater than 5 kts.

On‐board energy is provided by two packs of ZIPPY Flightmax 5000 mAh 6S1P LiPo batteries. This solution assures about 220 Wh of total on‐board energy. Taking into account the efficiency of the propulsion system, the continuous operation at 1.5 m/s (3 kts) for 20 min (resulting in range of 2 km) should require about 100 Wh of energy, leaving 120 Wh for electronics and com‐ munications, which consume about 10 Wh. The battery pack is enclosed in a watertight box that is located in the bow compartment. This compartment also houses another watertight box with the on‐board computer, navigation sensors and communications equipment. The bow compartment is also watertight assuring a double protection for electronics and batteries.

Navigation sensors include a PNI Trax AHRS and an Ublox Neo 6P GPS. Trax AHRS is a low‐ power and low‐cost attitude and heading reference system with a static heading accuracy of 0.2° and an overall accuracy better than 2° in the presence of magnetic distortions. NEO 6P is a low cost GPS receiver that operates at 10 Hz, outputs raw data, being supported by RTKLIB, an open source library that implements differential and real time kinematic corrections using small size and inexpensive receivers.

Communications with a control station are assured by a long‐range Wi‐Fi link that establishes a wide band link over distances above 1 km (depending on height of the shore station antenna over the water surface and on the wave conditions).

**Figure 5.** Unmanned capsule (source: ICARUS).

A video camera is also installed on the UCAP. A video stream is fed to the control station for possible assessment of victim conditions when the UCAP is close to them, as show in **Figure 6**.

Besides the described items, on‐board electronics also includes a load balancing and protec‐ tion system for the batteries, the motor controllers or the water jet motor and direction servo as well as triggering systems for the inflation of the raft. The interconnections between all on‐board systems are depicted in **Figure 7**.

The on‐board software is composed of several modules that communicate with each other using a message passing mechanism as shown in **Figure 8**.

These modules follow a hierarchical architecture similar to the one used in the other INESC TEC robotic systems.  On top of these, two major mod‐ ules are responsible for the navigation (real time estimation of the UCAP position, velocity and attitude) and for the control (execution of manoeuvres and other high level behaviours).

The navigation module processes data from the GPS and inertial measurement unit (IMU) sys‐ tems. The GPS system provides the information about location and velocity. The IMU has incor‐ porated magnetometers, accelerometers and gyroscopes, providing information about yaw, pitch and roll states, acceleration and rotational movements decomposed. A data fusion algorithm is used to estimate the position of the capsule whenever the GPS receiver loses track, possibly due to excessive roll or pitch caused by stronger waves. At the same time, the inertial data are used to obtain updated information on the external disturbances, allowing a better characterization of the navigation environment.

The UCAP carries a lightweight life raft as the one presented in **Figure 9**. This life raft weights 8 kg (raft + full inflation bottle) and the overall volume before inflation is 13 dm3 .

**Figure 6.** Image taken from the unmanned capsule on‐board camera (source: ICARUS).

**Figure 7.** System architecture—electronics (source: ICARUS).

**Figure 8.** System architecture—software (source: ICARUS).

**Figure 9.** Inflated life raft (source: ICARUS).

A mobile computer running software for real‐time monitoring and control of unmanned capsules composes the operation console. A graphical interface (**Figure 10**) provides the operator with the most relevant data concerning the state of a UCAP and allows him to control its operation. A joystick can be optionally connected to the computer to simplify the interaction with the UCAP.

This console allows the operator to switch between different UCAP operating modes:

**Figure 10.** UCAP operation console (source: ICARUS).

Specific information about the UCAP that is displayed in the graphical interface includes

When the UCAP is in autonomous operation, further information concerning the status of such operation (distance to next waypoint, estimated time no next waypoint completion or distance to anchor point in anchor mode) is also provided to the operator.

A heartbeat mechanism is implemented between the UCAPs and operator console to support emergency behaviour of the UCAP in case of communication link failure.

#### **6. Unmanned capsule deployment system**

The deployment system consists of a mechanical structure and a release system that could be easily modified or redesigned to suit several carrier USVs (**Figure 11**). The structure consists of a ramp that allows the gravity to be the main force imposing forward motion to the UCAP during launch. It is made of anodized aluminium bars to keep the overall weight low and resist to corrosion due to salt water. When placed on the launching ramp, the UCAP sits on rubber rollers that allow the movement in the forward direction while constraining it in the transversal direction [7].

The release system is composed by an electric latching device and an electronic system required for its command. This system is housed in a watertight box and is composed by a microcon‐ troller, a power amplifier and a battery. The system is connected to the carrier USV communica‐ tions infrastructure so that it can receive a remote command to release the UCAP. This command can be issued by a command line or a graphical interface running on any Linux‐based device.

The deployment system can be easily integrated on a carrier USV (**Figure 12**), requiring only the following operations:

Mounting the UCAP on the deployment system is a simple operation that can be performed, whereas the carrier USV is moored next to pier (**Figure 13**). The UCAP can be directly mounted on the rollers on ramp with the help of a rubber boat.

**Figure 11.** UCAP deployment system installed on Roaz II USV (source: ICARUS).

**Figure 12.** Deployment system installed on the U‐RANGER USV (source: ICARUS).

**Figure 13.** Two UCAPs mounted on the deployment system installed on the U‐RANGER (source: ICARUS).

Upon mounting the UCAP on the ramp, it must be secured to the release mechanism. For that purpose, a rope with a metal attached to it is fastened to the stern of the UCAP; and when the UCAP is in place, that pin is attached to the latching device.

Afterwards, for releasing the UCAP, a simple command needs to be sent to microcontroller in the electronic box of the deployment system (**Figure 14**).

**Figure 14.** UCAP being launched from the U‐RANGER (source: ICARUS).

#### **7. Conclusions**

This chapter addresses the work performed within the scope of the ICARUS project on the development of complementary unmanned maritime systems technologies for search and rescue. This work aimed at delivering a set of tools that can act not only as a part of the ICARUS toolset, but can also be used independently. For one side, such developments con‐ sisted in endowing medium and large scale unmanned surface vehicles with augmented per‐ ception and autonomic capabilities so that they could perform search and rescue operations in complex environments with the presence of other vessels and victims on the water, report‐ ing back to the control stations situational awareness information. On the other hand, the con‐ cept of unmanned capsule, which is a small‐size platform able to carry a life raft and inflate it close to victims, was prototyped, and integration as payload of larger unmanned platforms was also addressed. These developments and their extensive validation in several field trials and demonstrations carried out along the project are therefore a relevant contribution for the real‐world deployment of robotics platforms in search and rescue operations, complementing the operation of traditional search and rescue teams.

### **Acknowledgements**

These developments had the invaluable contribution of a large number of researchers of the ICARUS partners directly related to the maritime platforms and also counted with rel‐ evant inputs from other members of the ICARUS team. The authors thank all of them for their support. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement number 285417.

#### **Author details**

Aníbal Matos1,2\*, Eduardo Silva1,3, José Almeida1,3, Alfredo Martins1,3, Hugo Ferreira1 , Bruno Ferreira1 , José Alves1,2, André Dias1,3, Stefano Fioravanti4 , Daniele Bertin5 and Victor Lobo6

\*Address all correspondence to<EMAIL_ADDRESS>
1 IINESC TEC – Instituto de Engenharia de Sistemas e Computadores, Tecnologia e Ciência, Rua Dr. Roberto Frias, Porto, Portugal

2 Faculdade de Engenharia da Universidade do Porto, Rua Dr. Roberto Frias, Porto, Portugal

3 Instituto de Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida, Porto, Portugal

4 NATO Science and Technology Organisation – Centre for Maritime Research and Experimentation, Viale San Bartolomeo, La Spezia (SP), Italy

5 Calzoni S.r.l., Via A. De Gasperi, Calderara di Reno (BO), Italy

6 Escola Naval, Rua Base Naval de Lisboa, Almada, Portugal

#### **References**


3.1.1. Granulometric Analysis

The soil used for making the bricks, according to its particle distribution, showed 57.1% sand, 24.3% clay and 18.6% silt fraction, which is classified as sandy clay soil according to the ABNT standard [44]. This soil is considered suitable for making bricks as recommended by [45], who state that soils with sandy characteristics are the most suitable for the confection of soil-cement bricks, and also by the ABCP [46]. Indeed, the results of the granulometric analysis of the soil indicated that 100% passed through the ABNT 4.8 mm sieve (n◦. 4) and 10% to 50% passed through the ABNT 0.075 mm sieve (n◦ 200), as observed in the granulometric curve presented in Figure 3.

**Figure 3.** Natural soil and PET waste granulometric analysis.

The particle size distribution of PET particles was 99.9% sandy, 85.5% coarse, 11.5% medium and 2.9% fine particles, as shown in Figure 3. Since PET did not present consistency limits nor hygroscopicity, it can be considered non-plastic and sandy, according to the ABNT standard [44].