Orazzhanova, L.K., Yashkarova, H.G., Issue, L.A. & Kudaibergenov, S.E. (2003). *Binary*

Ro, K.W., Chang, W.J., Kim, H., Koo, Y.M. & Hahn, J.H. (2003). *Capilary electrochromatography*

Serniuk, S.E. & Thomas, R.M. (1965). *Copolymerization of polar with nonpolar monomers in the*

Serniuk, S.E. & Thomas, R.M. (1966). *Copolymerization with monomer complexes*. (to Esso

Tazuka, S. & Okamura, S. (1966). *Effect of metal salts on polymerization. Part 1. Polymerization of*

Tazuka, S., Sato, N. & Shimizu, S. (1966). *Effects of metal salts on polymerization. Part II.*

Tazuka, S., Okamura, S. (1967). *Effects of metal salts on polymerization. Part III. Radical*

Tsuchide, E., Nishide, H. & Takeshita, M.. (1974). *Steric and electrostatic factors on the formation*

Varghese, S., Lele, A.K., Sirnivas, D. & Mashellear, R.A. (2001). *Role of hydrophobicity on structure of polymer- metal complexes*, J. Phys. Chem. B 105 (23), 5368-5373. Varvara, S., Muresane, L., Popescu, I.C. & Maurin, G. (2004). *Copper electrodeposition from*

*vinylpyridine initiated with cupric acetate*, J. Polym. Sci. 4 (1), 141-157.

*salts*, J. Polym. Sci., Part A-1: Polym.Chem. Ed. 5 (5), 1083-1099.

*copolymerization of methacrylic acid coordinated to Co(III) complex with sodium styrene-4-*

*and ternary polymer- strontium complexes and the capture of radioactive strontium-90 from the polluted soil of the semipalalinsk nuclear test site*, J. Appl. Polym. Sci. 87 (5),

*and preconcentration of neutral compounds on poly(dimethylsiloxane) microchips,*

*presence of a Friedal-Crafts and a free radical initiator.* (to Essa Research & Engineering

*Polymerization of vinylpyridine complexed with the group IIb metal salts,* J. Polym. Sci.,

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*and the structure of polymeric cobalt(III) complexes,* Die Makromol. Chemie 175 (8)

*sulfate electrolytes in the presence of hydroxyethylated-2-butyne-1, 4-diol,*

*complexes: supramolecular modeling for determination and identification the band length in novel polymer complexes from their infrared spectra*, J. Appl. Organomet. Chem. 20,

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**8**

**Dermatological Application of PAMAM –**

Stoichiometry of reaction describes the quantitative relationships among substances as they participate in chemical reactions. Furthermore the term stoichiometry is used to describe the quantitative relationship among elements in compounds. This is also valid in the quantitative description of so-called weak complexes formed between molecules through non-covalent and non-ionic interactions. They generally arise from hydrogen bonding or Van der Waals interactions and are very common in biological chemistry, like in the case of enzyme-substrate, enzyme-inhibitor, or enzyme-coenzyme complexes. In less specific systems, stoichiometry may be referred to the number of small molecules interacting with one macromolecule to form complex structure. Depending on the interaction type between the components of the complex they are often desribed as host (macromolecule, H) – guest (small molecule, g) complexes. The host-guest complexes can sometimes be isolated as compounds of defined strochiometry, however their dissolution always leads to establishing an equilibrium between complexes and free guest, according

Hgn ⇔ Hgn-1 + g ⇔ Hgn-2 + 2g ⇔ Hgn-3 + 3g ……. ⇔ H + ng (1)

This weak interaction between H and g leads to the formation of multiple complexes Hgn, with n = 1, 2, 3, …. n, depending on g concentration, which is in excess compared to H one. Its larger amount is necessary to determine nmax, i.e. the maximum amount of guest

**1. Introduction**

to the scheme:

described by overall formation constant:

**Vitamin Bioconjugates and Host-Guest**

**Complexes – Vitamin C Case Study**

Stanisław Wołowiec1, Marek Laskowski1, Barbara Laskowska1, Agnieszka Magoń2, Bogdan Mysliwiec2 and Marek Pyda2

*University of Information Technology and Management, 2Faculty of Chemistry, Rzeszów University of Technology,*

K = [Hgn]/[H][g]n (2)

*1Department of Cosmetology,*

*Rzeszów Poland*

## **Dermatological Application of PAMAM – Vitamin Bioconjugates and Host-Guest Complexes – Vitamin C Case Study**

Stanisław Wołowiec1, Marek Laskowski1, Barbara Laskowska1, Agnieszka Magoń2, Bogdan Mysliwiec2 and Marek Pyda2 *1Department of Cosmetology, University of Information Technology and Management, 2Faculty of Chemistry, Rzeszów University of Technology, Rzeszów Poland*

### **1. Introduction**

194 Stoichiometry and Research – The Importance of Quantity in Biomedicine

Wertz, D.L. & Tyroll, L. (1974). *The coordination of Cu(II) in a nearly saturated solution of CuCl2*

Zubov, V.P., Lachinov, N.B., Golubov, V.B., Kilikova, V.F., Kabanov, V.A., Polak, L.S. &

Kargin, V.A. (1968). *The influence of complexing agents on the polymerization of vinyl*

*in hydrochloric acid,* J. Inorg. & Nucl. Chem. 36 (12), 3713-3717.

*and allyl monomers*, J. Polym. Sci., Part C 23 (1), 147-155.

Stoichiometry of reaction describes the quantitative relationships among substances as they participate in chemical reactions. Furthermore the term stoichiometry is used to describe the quantitative relationship among elements in compounds. This is also valid in the quantitative description of so-called weak complexes formed between molecules through non-covalent and non-ionic interactions. They generally arise from hydrogen bonding or Van der Waals interactions and are very common in biological chemistry, like in the case of enzyme-substrate, enzyme-inhibitor, or enzyme-coenzyme complexes. In less specific systems, stoichiometry may be referred to the number of small molecules interacting with one macromolecule to form complex structure. Depending on the interaction type between the components of the complex they are often desribed as host (macromolecule, H) – guest (small molecule, g) complexes. The host-guest complexes can sometimes be isolated as compounds of defined strochiometry, however their dissolution always leads to establishing an equilibrium between complexes and free guest, according to the scheme:

$$\mathrm{Hg}\_{\mathrm{n}} \Leftrightarrow \mathrm{Hg}\_{\mathrm{p}\cdot 1} + \mathrm{g} \Leftrightarrow \mathrm{Hg}\_{\mathrm{p}\cdot 2} + 2\mathrm{g} \Leftrightarrow \mathrm{Hg}\_{\mathrm{p}\cdot 3} + 3\mathrm{g} \cdot \dots \dots \Leftrightarrow \mathrm{H} + \mathrm{ng} \tag{1}$$

described by overall formation constant:

$$\mathbf{K} = [\mathbf{H} \mathbf{g}\_n] / [\mathbf{H}] [\mathbf{g}]^n \tag{2}$$

This weak interaction between H and g leads to the formation of multiple complexes Hgn, with n = 1, 2, 3, …. n, depending on g concentration, which is in excess compared to H one. Its larger amount is necessary to determine nmax, i.e. the maximum amount of guest

Dermatological Application of PAMAM –

**N N**

**O OMe**

**O**

**N H N**

**NH**

**O**

**N**

**O**

**2NH NH2**

**OMe**

**O OMe**

**O**

**O**

**MeO**

to G1.5, G2, G2,5, G3 and so on.

**MeO**

**2.2 Medical applications of PAMAM dendrimers**

**O**

**MeO**

**O**

**O**

**G-0.5**

**MeO**

**MeO**

Vitamin Bioconjugates and Host-Guest Complexes – Vitamin C Case Study 197

**N N**

**O**

**O N H**

> **O OMe**

**NH2**

**G0**

**O**

**NH**

**NH2**

**O**

**O N H** **N**

**O**

**G0.5**

**OMe**

**OMe**

**O**

**O**

**N H 2NH**

**2NH NH2**

**N N**

**O**

**NH**

**O N**

**O**

**OMe**

Scheme 1. The scheme of synthesis of PAMAM dendrimers on ethylenediamine (en) core. Addition of methyl acrylate to en results in formation of -0.5 half-generation PAMAM dendrimer (G-0.5) with four methyl ester groups on surface. The condensation of G-0.5 with four en equivalents leads to full-generation PAMAM dendrimer (G0). Further addition of methyl acrylate gives G0.5 PAMAM dendrimer, which upon next condensation with eight en equivalents results in obtaining full generation PAMAM dendrimer G1 (not shown). Repeated sequence of addition/condensation protocol leads

PAMAM dendrimers are relatively low toxic (Jain et al., 2010, Mukherjee et al., 2010, Kolhatkar et al., 2007, Hans & Lowman, 2002). Therefore they are extensively studied as drug carriers. There are two strategies commonly applied: 1) diffusion of prodrug covalently bound to the dendrimer, and 2) diffusion of drug encapsulated in the dendrimer. Thus, the PAMAM dendrimers act as solubilizers for anti-inflamatory drugs *ketoprofen* (Yiyun et al., 2005), *ibuprofen* (Milhem et al., 2000) and *indomethacin* (Chauhan et al., 2003) and promote

**NH**

**NH2**

**MeO OMe**

molecules which are able to interact with one host macromolecule. Thus the nmax is often referred to as host-guest complex stoichiometry or host capacity in relation to specific guest.

Commonly employed molecular complex is the one used in iodometric titration, where the equivalent point is determined by the deeply blue color of starch (macromolecular host) and iodine (guest) complex disappearance with the iodine completely consumed by thiosulfate (Rundle & Frank, 1943).

Starch is a glucose polymer containing mainly α(1-4) glycosidic bonds with an amylose moiety forming polyiodide complexes with maximum absorbance at 620 nm. Being starch a polydisperse polymer, the equilibrium constant for the blue iodine complex formation is actually the averaged value (Saenger, 1984).

More adequate formation constants can be determined for monodisperse macromolecules, such as dendritic polymers. The dendrimer chemistry developed very fast over the last two decades, and it has been the subject of many reviews due to dendrimers numerous applications, such as: nanomaterials for molecular electronics, photonics, sensors (Astruc et al., 2010, Lo & Burn, 2007) and nanomedicine (D'Emanuele & Attwood, 2005, Caruthers et al., 2007). Polyethyleneimine (PEI), polyester (PES) and polyamidoamine (PAMAM) dendrimers invented in Frechet's and Tomalia's group macromolecules own a signifficant position among dendrimeric molecules (Frechet et al., 1995, Tomalia , 2005a and b).

### **2. Polyamidoamine dendrimers (PAMAM)**

### **2.1 General characteristics of PAMAM dendrimers**

The dendritic molecules are globular shaped monodisperse objects. The polyamidoamine dendrimers (PAMAM) were synthesized in Tomalia's group (Tomalia et al., 2003, Tomalia, 2005). The PAMAM dendrimers of full generation posses several surface amine groups which make them perfectly soluble in water. They can be obtained by alternate addition of methyl acrylate to amine (in our study ethylenediamine has been used as starting diamine core) resulting in doubling the surface methyl propionate groups, followed by condensation of ester groups with diamine (ethylenediamine being the best choice). The latter step introduces further amine function on surface. Thus the full generation (Gn) PAMAM dendrimers posses: 8, 16, 32, and 64 surface amine groups in G1, G2, G3, and G4, respectively. The synthetic pattern is shown in Scheme 1. The hydrodynamic diameters of full generation PAMAM dendrimers are: 1.5, 2.2, 2.9, 3.6, and 4.5 nm for G0, G1, G2, G3, and G4, respectively. The surface structure of full generation dendrimers can be widely modified by surface amine groups conversion, resulting in change in their solubility and permeability properties (Jevprasesphant et al., 2003, Imae et al., 2000). The PAMAM dendrimers are molecules with inner cavities, which are able to absorb small molecules to form host-guest complexes. During the synthesis of PAMAM dendrimers in methanol, the final removal of this solvent from the dendrimer spherical macromolecule requires prolonged vacuum evaporation. PAMAM dendrimers ability to encapsulate hydrophobic guests was extensively used as a strategy for promoting water insoluble drugs transportation into tissues and cells (vide infra).

molecules which are able to interact with one host macromolecule. Thus the nmax is often referred to as host-guest complex stoichiometry or host capacity in relation to specific guest. Commonly employed molecular complex is the one used in iodometric titration, where the equivalent point is determined by the deeply blue color of starch (macromolecular host) and iodine (guest) complex disappearance with the iodine completely consumed by thiosulfate

Starch is a glucose polymer containing mainly α(1-4) glycosidic bonds with an amylose moiety forming polyiodide complexes with maximum absorbance at 620 nm. Being starch a polydisperse polymer, the equilibrium constant for the blue iodine complex formation is

More adequate formation constants can be determined for monodisperse macromolecules, such as dendritic polymers. The dendrimer chemistry developed very fast over the last two decades, and it has been the subject of many reviews due to dendrimers numerous applications, such as: nanomaterials for molecular electronics, photonics, sensors (Astruc et al., 2010, Lo & Burn, 2007) and nanomedicine (D'Emanuele & Attwood, 2005, Caruthers et al., 2007). Polyethyleneimine (PEI), polyester (PES) and polyamidoamine (PAMAM) dendrimers invented in Frechet's and Tomalia's group macromolecules own a signifficant position among dendrimeric molecules (Frechet et al., 1995, Tomalia , 2005a

The dendritic molecules are globular shaped monodisperse objects. The polyamidoamine dendrimers (PAMAM) were synthesized in Tomalia's group (Tomalia et al., 2003, Tomalia, 2005). The PAMAM dendrimers of full generation posses several surface amine groups which make them perfectly soluble in water. They can be obtained by alternate addition of methyl acrylate to amine (in our study ethylenediamine has been used as starting diamine core) resulting in doubling the surface methyl propionate groups, followed by condensation of ester groups with diamine (ethylenediamine being the best choice). The latter step introduces further amine function on surface. Thus the full generation (Gn) PAMAM dendrimers posses: 8, 16, 32, and 64 surface amine groups in G1, G2, G3, and G4, respectively. The synthetic pattern is shown in Scheme 1. The hydrodynamic diameters of full generation PAMAM dendrimers are: 1.5, 2.2, 2.9, 3.6, and 4.5 nm for G0, G1, G2, G3, and G4, respectively. The surface structure of full generation dendrimers can be widely modified by surface amine groups conversion, resulting in change in their solubility and permeability properties (Jevprasesphant et al., 2003, Imae et al., 2000). The PAMAM dendrimers are molecules with inner cavities, which are able to absorb small molecules to form host-guest complexes. During the synthesis of PAMAM dendrimers in methanol, the final removal of this solvent from the dendrimer spherical macromolecule requires prolonged vacuum evaporation. PAMAM dendrimers ability to encapsulate hydrophobic guests was extensively used as a strategy for promoting water insoluble drugs transportation into

(Rundle & Frank, 1943).

and b).

actually the averaged value (Saenger, 1984).

**2. Polyamidoamine dendrimers (PAMAM)**

tissues and cells (vide infra).

**2.1 General characteristics of PAMAM dendrimers**

Scheme 1. The scheme of synthesis of PAMAM dendrimers on ethylenediamine (en) core. Addition of methyl acrylate to en results in formation of -0.5 half-generation PAMAM dendrimer (G-0.5) with four methyl ester groups on surface. The condensation of G-0.5 with four en equivalents leads to full-generation PAMAM dendrimer (G0). Further addition of methyl acrylate gives G0.5 PAMAM dendrimer, which upon next condensation with eight en equivalents results in obtaining full generation PAMAM dendrimer G1 (not shown). Repeated sequence of addition/condensation protocol leads to G1.5, G2, G2,5, G3 and so on.

### **2.2 Medical applications of PAMAM dendrimers**

PAMAM dendrimers are relatively low toxic (Jain et al., 2010, Mukherjee et al., 2010, Kolhatkar et al., 2007, Hans & Lowman, 2002). Therefore they are extensively studied as drug carriers. There are two strategies commonly applied: 1) diffusion of prodrug covalently bound to the dendrimer, and 2) diffusion of drug encapsulated in the dendrimer. Thus, the PAMAM dendrimers act as solubilizers for anti-inflamatory drugs *ketoprofen* (Yiyun et al., 2005), *ibuprofen* (Milhem et al., 2000) and *indomethacin* (Chauhan et al., 2003) and promote

Dermatological Application of PAMAM –

cosmetic creams.

**3.1 Materials and methods**

used without further purification.

**3.1.2 Syntheses of PAMAM dendrimers**

based on 1-D and 2-D NMR experiments.

**3.1.1 Reagents**

Vitamin Bioconjugates and Host-Guest Complexes – Vitamin C Case Study 199

matrix of intercellular lipid due to its low water content. This also demonstrated that the rapid permeation of vitamin C when released from delivering formulation is not necessarily a cosmetic target. Macromolecular carriers can be used for controlling the transdermal diffusion rate of cosmetic ingredients; polyamidoamine (PAMAM) dendrimers, widely used as transdermal carriers for drugs have been shown to be amongst the best candidates (Svenson, 2009). For water-insoluble vitamins, like A and E, they can serve as solubilizers, while for water soluble vitamins, like ascorbic acid (**1**), they might allow to increase the skin load. In such a case the PAMAM dendrimers could be used as valuable additives for

Here we present the results on simple host-guest chemistry between PAMAM dendrimers and **1** in solution and in neat dendrimers **G2,5**, **G3**, **G3,5**, and **G4** studied by the 1H NMR and differential scanning calorimetry (DSC) methods in order to establish the proper stochiometry of host-guest complexes available for cream formulation. The preliminary *in vitro* studies on diffusion of **1** absorbed in PAMAM dendrimers through artificial model

**O**

**1**

**OH O**

**OH OH**

**1**

Ascorbic acid (vitamin C, **1**, MW = 176.12 g/mol, m.p. = 190 - 194°C with decomposition) was used as received. All solvents and reagents were of reagent grade purity (Aldrich) and

PAMAM dendrimers of generation 2, 2,5, 3, 3,5, and 4 (**G2**, **G2,5**, **G3**, **G3,5**, and **G4**, respectively) on ethylenediamine core were synthesized according to the published method by alternate addition of methyl acrylate to **Gn** and condensation of ethylenediamine with **Gn,5** [Tomalia et al., 2003]. The dendrimers were characterized by the 1H and 13C NMR spectra in deuterium oxide and in methanol-*d*4 using standard 1-D and 2-D COSY, HMBC, and HSQC methods with 500 MHz Bruker UltraShield spectrometer to confirm their purity.

The solubility of **1** in methanol-*d4*, estimated by reference chloroform addition into the saturated solution is 0.36 mol dm-3. 1H and 13C resonances assignement has been performed

**3.1.3 Solubilization of 1 in methanol containing host dendrimers**

**3 2**

membrane (polyvinydifluoride, PVDF) and pig ear skin (PES) were also performed.

**OH**

**H**

**4 5 6**

their prolonged release both *in vitro* and *in vivo* (Na et al., 2006, Kolhe et al., 2003). Similar concept was used to increase solubility and uptake flux of several anticancer drugs, like *methoxyestradiol* (Wang et al., 2011), *adriamycin* and *methotrexate* (Kojima et al., 2000), a plant alkaloid *camptothecin* (Cheng et al., 2008), *doxorubicidin* (Papagiannaros et al., 2005), and *dimethoxycurcumin* (Markatou et al. 2007). Moreover, *nifedipine* - a calcium channel blocking agent (Devarakonda et al., 2004, 2005), a loop diuretic *furosemide* (Devarakonda et al., 2007, antipsychotic drug *risperidone* (Prieto et al., 2011), a hypolipidemic drug (Kulhari et al., 2011), antibiotic *quinolones* (Cheng et al., 2007), antibacterial *sulfamethoxazole* (Ma et al., 2007), as well as ophtalmic drugs *pilocarpine nitrate* and *tropicamide* (Vandamme & Brobeck, 2005) were also supported by PAMAM dendrimers as carrier. Surprisingly PAMAM – drug complexes were proven to be effectively transdermal increasing the flux of *5-fluorouracil* - a drug for treatment of psoriasis, premalignant and malignant skin conditions (Venuganti & Perumal, 2008, Bhadra et al., 2003) of dermatologically important vitamins, like *nicotinic acid* Cheng & Xu, 2005) and *riboflavin* (Filipowicz & Wołowiec, 2011) as well as of *psoralene* – the photosensitizer used for treatment of psoriasis (Borowska et al., 2010).

A more advanced strategy is based on a covalent bonding of the prodrug, bioconjugates being extremely promising for site-directed therapy.  Similar studies are currently being performed in our laboratory to clarify the skin penetration of vitamins supported by PAMAM dendrimers. In some cases PAMAM dendrimers act as solubilizers and enhance the skin load, while in other cases the vitamin flux decreases. Here we present the latest results on vitamin C permeation in the presence of PAMAM dendrimers.

### **3. PAMAM dendrimers as carriers for vitamin C**

Ascorbic acid (vitamin C) is an essential nutrient protecting living tissues from oxidation processes by free radicals and reactive oxygen derived species. It is also an important cofactor in collagen synthesis, hence present in most cosmetic products. The bioavailability of topically applied vitamin C is very high compared with other dermatologically important vitamins and depends on the specific delivery system, like o/w, w/o emulsions and hydrogels (Rozman et al., 2009) and liposomes, niosomes or solid lipid nanoparticles as dispergents (Kaur et al., 2007) as well as microemulsions (Kogan and Garbi, 2006). Due to its instability vitamin C is usually present in cosmetics as ester derivatives which are thermally and oxidatively more stable. Besides they also promote diffusion through epidermis. Though ascorbic palmitate is generally used, recently some other esters (Shibayama et al., 2008, Kumano et al., 1998, Tai et al., 2004) and even tandem retinyl ascorbate (Abdulmayed and Herda, 2004) were synthesized and their skin bioavailability tested.

The permeation rate through whole mouse skin was estimated as 3.43 (± 0.74) μg/cm2/h (Lee and Tojo, 1998) assuming that the least permeable barrier of *stratum corneum* was a matrix of intercellular lipid due to its low water content. This also demonstrated that the rapid permeation of vitamin C when released from delivering formulation is not necessarily a cosmetic target. Macromolecular carriers can be used for controlling the transdermal diffusion rate of cosmetic ingredients; polyamidoamine (PAMAM) dendrimers, widely used as transdermal carriers for drugs have been shown to be amongst the best candidates (Svenson, 2009). For water-insoluble vitamins, like A and E, they can serve as solubilizers, while for water soluble vitamins, like ascorbic acid (**1**), they might allow to increase the skin load. In such a case the PAMAM dendrimers could be used as valuable additives for cosmetic creams.

Here we present the results on simple host-guest chemistry between PAMAM dendrimers and **1** in solution and in neat dendrimers **G2,5**, **G3**, **G3,5**, and **G4** studied by the 1H NMR and differential scanning calorimetry (DSC) methods in order to establish the proper stochiometry of host-guest complexes available for cream formulation. The preliminary *in vitro* studies on diffusion of **1** absorbed in PAMAM dendrimers through artificial model membrane (polyvinydifluoride, PVDF) and pig ear skin (PES) were also performed.

### **3.1 Materials and methods**

### **3.1.1 Reagents**

198 Stoichiometry and Research – The Importance of Quantity in Biomedicine

their prolonged release both *in vitro* and *in vivo* (Na et al., 2006, Kolhe et al., 2003). Similar concept was used to increase solubility and uptake flux of several anticancer drugs, like *methoxyestradiol* (Wang et al., 2011), *adriamycin* and *methotrexate* (Kojima et al., 2000), a plant alkaloid *camptothecin* (Cheng et al., 2008), *doxorubicidin* (Papagiannaros et al., 2005), and *dimethoxycurcumin* (Markatou et al. 2007). Moreover, *nifedipine* - a calcium channel blocking agent (Devarakonda et al., 2004, 2005), a loop diuretic *furosemide* (Devarakonda et al., 2007, antipsychotic drug *risperidone* (Prieto et al., 2011), a hypolipidemic drug (Kulhari et al., 2011), antibiotic *quinolones* (Cheng et al., 2007), antibacterial *sulfamethoxazole* (Ma et al., 2007), as well as ophtalmic drugs *pilocarpine nitrate* and *tropicamide* (Vandamme & Brobeck, 2005) were also supported by PAMAM dendrimers as carrier. Surprisingly PAMAM – drug complexes were proven to be effectively transdermal increasing the flux of *5-fluorouracil* - a drug for treatment of psoriasis, premalignant and malignant skin conditions (Venuganti & Perumal, 2008, Bhadra et al., 2003) of dermatologically important vitamins, like *nicotinic acid* Cheng & Xu, 2005) and *riboflavin* (Filipowicz & Wołowiec, 2011) as well as of *psoralene* – the

A more advanced strategy is based on a covalent bonding of the prodrug, bioconjugates being extremely promising for site-directed therapy.  Similar studies are currently being performed in our laboratory to clarify the skin penetration of vitamins supported by PAMAM dendrimers. In some cases PAMAM dendrimers act as solubilizers and enhance the skin load, while in other cases the vitamin flux decreases. Here we present the latest results on vitamin C permeation in the presence of PAMAM

Ascorbic acid (vitamin C) is an essential nutrient protecting living tissues from oxidation processes by free radicals and reactive oxygen derived species. It is also an important cofactor in collagen synthesis, hence present in most cosmetic products. The bioavailability of topically applied vitamin C is very high compared with other dermatologically important vitamins and depends on the specific delivery system, like o/w, w/o emulsions and hydrogels (Rozman et al., 2009) and liposomes, niosomes or solid lipid nanoparticles as dispergents (Kaur et al., 2007) as well as microemulsions (Kogan and Garbi, 2006). Due to its instability vitamin C is usually present in cosmetics as ester derivatives which are thermally and oxidatively more stable. Besides they also promote diffusion through epidermis. Though ascorbic palmitate is generally used, recently some other esters (Shibayama et al., 2008, Kumano et al., 1998, Tai et al., 2004) and even tandem retinyl ascorbate (Abdulmayed

The permeation rate through whole mouse skin was estimated as 3.43 (± 0.74) μg/cm2/h (Lee and Tojo, 1998) assuming that the least permeable barrier of *stratum corneum* was a

photosensitizer used for treatment of psoriasis (Borowska et al., 2010).

**3. PAMAM dendrimers as carriers for vitamin C**

and Herda, 2004) were synthesized and their skin bioavailability tested.

dendrimers.

Ascorbic acid (vitamin C, **1**, MW = 176.12 g/mol, m.p. = 190 - 194°C with decomposition) was used as received. All solvents and reagents were of reagent grade purity (Aldrich) and used without further purification.

### **3.1.2 Syntheses of PAMAM dendrimers**

PAMAM dendrimers of generation 2, 2,5, 3, 3,5, and 4 (**G2**, **G2,5**, **G3**, **G3,5**, and **G4**, respectively) on ethylenediamine core were synthesized according to the published method by alternate addition of methyl acrylate to **Gn** and condensation of ethylenediamine with **Gn,5** [Tomalia et al., 2003]. The dendrimers were characterized by the 1H and 13C NMR spectra in deuterium oxide and in methanol-*d*4 using standard 1-D and 2-D COSY, HMBC, and HSQC methods with 500 MHz Bruker UltraShield spectrometer to confirm their purity.

### **3.1.3 Solubilization of 1 in methanol containing host dendrimers**

The solubility of **1** in methanol-*d4*, estimated by reference chloroform addition into the saturated solution is 0.36 mol dm-3. 1H and 13C resonances assignement has been performed based on 1-D and 2-D NMR experiments.

Dermatological Application of PAMAM –

**[vit.C] [mol dm-3**

**]**

**0,30 0,35 0,40 0,45 0,50 0,55 0,60 0,65 0,70 0,75 0,80**

> **1 : Gn ratio**

> > 6:1 250 7:1 225

**3.1.5** *In vitro* **permeation of Gn-1 and Gn,5-1 complexes**

G3.5.

Vitamin Bioconjugates and Host-Guest Complexes – Vitamin C Case Study 201

**0,0000 0,0005 0,0010 0,0015 0,0020 0,0025 0,0030**

**G2,5 G3 G3,5 G4**

Fig. 2. The dependence of solubility of 1 in methanol containing variable concentration of

0:1 229 253 224 248 1:1 243 265 240 263 2:1 243 268 251 269 3:1 247 267 255 259 4:1 246 257 224 249 5:1 244 254 248

Table 1. Glass transition temperatures [K] for Gn dendrimers and mixtures of Gn and 1.

Permeation of **Gn**-**1** and **Gn,5**-**1** complexes was studied using Franz diffusion assembly (Thermo Scientific (UK) model DC 600 equipped with 6 cm3 acceptor compartments). The o/w emulsion was used as donor. The emulsion was prepared using cetearyl alcohol (1.5 g), Brij 72 (1.2 g), Brij 58 (0.3 g) as emulsifiers, vaseline (5.0 g), stearine (0.5 g), glycerin (1.5 g) and water (40.0 g). The samples containing **Gn**-**1** complexes were prepared on 1 g scale by dissolving **1 (***ca* 5 mg) and **Gn** (*ca* 50 mg) in 1 g of the emulsion. Preliminary samples were obtained by dissolving the host-guest complexes **Gn**-**1** (prepared by mixing **1** and **Gn** in methanol followed by vacuum evaporation) in emulsion on the same scale. No difference in the permeation studies were noticed related to the protocols. For the analysis ca 250 mg samples were mounted over commercial polyvinydifluoride membrane (PVDF, 0.125 mm thickness) or prepared pig ear skin membrane (PES, 0.55 mm thickness), with 0.067 M phosphate buffer pH = 7.4 : ethanol 7:3 v/v. as receptor medium. The progress of diffusion was monitored spectrophotometrically at 264 nm using the extinction coefficient calculated for the solution of **1** in receptor solution (1.1⋅104, Figure 3). The receiving solution was

**[C] = C0**

**R = 0.999**

**C0**

 **+ B [G3,5]**

 **= 0.35 (0.01); B = 160 (12)**

**[G3,5] [mol dm-3]**

1H NMR (chemical shift [ppm], intensity, multiplicity, assignment): 4.88 ([1H], d, H4); 3.90 ([1H], d of t, H5); 3.68 ([2H], d, H6); 13C NMR: 172.1 (C1); 153.4 (C3); 118.5 (C2); 75.5 (C4); 69.2 (C5); 62.2 (C6).

The solubilization of **1** in presence of dendrimers was studied by straight addition of solid **1** into 700 μL of **G3**, **G4**, **G2,5** or **G3,5** solutions at variable concentration in methanol-*d*4 after added a measured amount of chloroform as quantitative reference. The H4 doublet of (**1**) shifted from 4.88 to 4.73 ppm upon dissolving in solution containing **G3** or **G4** dendrimers. Partial precipitation of the complex occurred when added amount of **1** added reached **1**:**G3** and **1**:**G4** ratio equal to 3:1. The analysis of separated solid indicated that the composition of water-soluble solid complex was 3:1, independently on dendrimers concentration. Solubilization of **1** in methanol-*d*4 in presence of **G2,5** or **G3,5** showed no complexes precipitation within studied range of dendrimer concentration (up to 0.003 mol⋅dm-3); the solubility of **1** depending linearly on it.

 Linear regression of **1** versus **G2,5** and **G3,5** concentrations are shown in Figure 1 and 2, respectively. The proton resonances in the 1H NMR spectrum of **1** in solutions containing **G2,5** or **G3,5** were not shifted compared with **1** in pure methanol-*d*4.

### **3.1.4 Differential scanning calorimetric studies on PAMAM-1 stoichiometry**

Glass transition temperature Tg was examined using heat-flux differential scanning calorimeter DSC, Q1000™ from TA Instruments, Inc., equipped with a mechanical refrigerator from temperatures 183.15 K (-90°C ) to 393.15 K (120°C) (dry nitrogen gas with a flow rate of 50 cm3⋅min-1 was purged through the DSC cell in the instrument and cooling was accomplished with a refrigerated cooling system). Samples of neat **G2,5**, **G3**, **G3,5**, and **G4** dendrimers were examined as well as host-guest complexes prepared by dissolving **Gn** or **Gn,5** in methanol followed by addition of measured amount of **1** and extensive vacuum evaporation. The oily samples of **1** and **Gn** or **Gn,5** exhibited a higher Tg value than the neat dendrimers (Table 1) within **1**:**PAMAM** ratio 6:1 for **G2,5** and 3:1 for other generations of PAMAM dendrimer. The original Tg of pure dendrimers was recovered once exceeded these molar ratios, with a subsequent lost of the sample homogeneity.

Fig. 1. The dependence of solubility of 1 in methanol containing variable concentration of G2.5.

1H NMR (chemical shift [ppm], intensity, multiplicity, assignment): 4.88 ([1H], d, H4); 3.90 ([1H], d of t, H5); 3.68 ([2H], d, H6); 13C NMR: 172.1 (C1); 153.4 (C3); 118.5 (C2); 75.5 (C4); 69.2

The solubilization of **1** in presence of dendrimers was studied by straight addition of solid **1** into 700 μL of **G3**, **G4**, **G2,5** or **G3,5** solutions at variable concentration in methanol-*d*4 after added a measured amount of chloroform as quantitative reference. The H4 doublet of (**1**) shifted from 4.88 to 4.73 ppm upon dissolving in solution containing **G3** or **G4** dendrimers. Partial precipitation of the complex occurred when added amount of **1** added reached **1**:**G3** and **1**:**G4** ratio equal to 3:1. The analysis of separated solid indicated that the composition of water-soluble solid complex was 3:1, independently on dendrimers concentration. Solubilization of **1** in methanol-*d*4 in presence of **G2,5** or **G3,5** showed no complexes precipitation within studied range of dendrimer concentration (up to 0.003 mol⋅dm-3); the

 Linear regression of **1** versus **G2,5** and **G3,5** concentrations are shown in Figure 1 and 2, respectively. The proton resonances in the 1H NMR spectrum of **1** in solutions containing

Glass transition temperature Tg was examined using heat-flux differential scanning calorimeter DSC, Q1000™ from TA Instruments, Inc., equipped with a mechanical refrigerator from temperatures 183.15 K (-90°C ) to 393.15 K (120°C) (dry nitrogen gas with a flow rate of 50 cm3⋅min-1 was purged through the DSC cell in the instrument and cooling was accomplished with a refrigerated cooling system). Samples of neat **G2,5**, **G3**, **G3,5**, and **G4** dendrimers were examined as well as host-guest complexes prepared by dissolving **Gn** or **Gn,5** in methanol followed by addition of measured amount of **1** and extensive vacuum evaporation. The oily samples of **1** and **Gn** or **Gn,5** exhibited a higher Tg value than the neat dendrimers (Table 1) within **1**:**PAMAM** ratio 6:1 for **G2,5** and 3:1 for other generations of PAMAM dendrimer. The original Tg of pure dendrimers was recovered once exceeded these

**0,0000 0,0005 0,0010 0,0015 0,0020 0,0025 0,0030**

Fig. 1. The dependence of solubility of 1 in methanol containing variable concentration of G2.5.

**[C] = C0**

**R = 0.990**

**C0**

 **+ B [G2,5]**

**[G2,5] [mol dm-3**

**]**

 **= 0.36 (0.02); B = 144 (12)**

**G2,5** or **G3,5** were not shifted compared with **1** in pure methanol-*d*4.

molar ratios, with a subsequent lost of the sample homogeneity.

**0,30 0,35 0,40 0,45 0,50 0,55 0,60 0,65 0,70 0,75 0,80**

**[vit.C] [mol dm**

**-3**

**]**

**3.1.4 Differential scanning calorimetric studies on PAMAM-1 stoichiometry**

(C5); 62.2 (C6).

solubility of **1** depending linearly on it.

Fig. 2. The dependence of solubility of 1 in methanol containing variable concentration of G3.5.

Table 1. Glass transition temperatures [K] for Gn dendrimers and mixtures of Gn and 1.

### **3.1.5** *In vitro* **permeation of Gn-1 and Gn,5-1 complexes**

Permeation of **Gn**-**1** and **Gn,5**-**1** complexes was studied using Franz diffusion assembly (Thermo Scientific (UK) model DC 600 equipped with 6 cm3 acceptor compartments). The o/w emulsion was used as donor. The emulsion was prepared using cetearyl alcohol (1.5 g), Brij 72 (1.2 g), Brij 58 (0.3 g) as emulsifiers, vaseline (5.0 g), stearine (0.5 g), glycerin (1.5 g) and water (40.0 g). The samples containing **Gn**-**1** complexes were prepared on 1 g scale by dissolving **1 (***ca* 5 mg) and **Gn** (*ca* 50 mg) in 1 g of the emulsion. Preliminary samples were obtained by dissolving the host-guest complexes **Gn**-**1** (prepared by mixing **1** and **Gn** in methanol followed by vacuum evaporation) in emulsion on the same scale. No difference in the permeation studies were noticed related to the protocols. For the analysis ca 250 mg samples were mounted over commercial polyvinydifluoride membrane (PVDF, 0.125 mm thickness) or prepared pig ear skin membrane (PES, 0.55 mm thickness), with 0.067 M phosphate buffer pH = 7.4 : ethanol 7:3 v/v. as receptor medium. The progress of diffusion was monitored spectrophotometrically at 264 nm using the extinction coefficient calculated for the solution of **1** in receptor solution (1.1⋅104, Figure 3). The receiving solution was

Dermatological Application of PAMAM –

experiments on skin-model membranes (see below).

**dendrimers**

3:1; 4:1.

**3.2.3 Permeation studies**

Vitamin Bioconjugates and Host-Guest Complexes – Vitamin C Case Study 203

Recently we have used DSC method to evaluate the stoichiometry of host-guest complexes between PAMAM dendrimers and *8-methoxypsoralene* (Borowska et al., 2010). Water insoluble host formed oily host-guest complexes which revealed higher temperature of glass transition (Tg) than PAMAM dendrimers. When the guest amount was increased above the host maximum capacity, the separation of PAMAM dendrimers and guest took place, followed by Tg returning to its original value. Similar phenomenon was observed in all cases studied here. However, the results on Tg were slightly surprising, because the limit of encapsulation for dendrimers **G3**, **G3,5** and **G4** was 3 molecules of **1** per macromolecule, i.e. lower in comparison with **G2,5**, for which 6 molecules of **1** per molecule of **G2,5**, showed increased value of Tg. However it confirms the 1H NMR results on solubilization in methanol, which also showed that **G2,5** interacts with more molecules of guest than larger hosts. This might be due to different types of interaction between host and **1**. Vitamin C, unlike hydrophobic *8-methoxypsoralene* is able to form ion-pair complex, this interaction prevailing in case of full-generation dendrimers **G3** and **G4**. The ion-pairs decompose when solvent is removed from solution upon preparation of neat samples. Besides, the larger dendrimers are used, the more dense are the cavities of host and self organization of vitamin C crystals prevails over weak intramolecular interaction within host-guest complex. Furthermore we noticed that homogeneous samples of host-guest complexes formed between PAMAM dendrimers and **1** can be obtained in every case when the stoichiometry is maintained as 3:1. These complexes were then used to perform transdermal permeability

Fig. 4. The DSC curves for **1** : **G4** mixtures at molar ratio (from bottom to top): 0:1; 1:1; 2:1;

The permeation experiments of **1** through PVDF and PES membranes using **1** dispersed in o/w emulsion indicated a typical change in the flux of **1**; after 0.5 hr induction time, the flux

rapidly grew finally stabilizing within 1.5 hour of experiment.

**3.2.2 Differential scanning calorimetric studies of neat complexes of 1 with**

stirred magnetically with 1000 rpm at 32°C. 10 ml aliquots of receptor solution were taken at 0.5 hour or longer time intervals and the receiver compartment was filled with 6 ml portion of a new receptor solution. The experiments were carried on until at least 10% of initial amount of **1** was received in receptor solution. The results were analyzed calculating the flux in [μmol⋅hour-1⋅cm-2]. The active area of membrane determined by size of the ring in Franz cell was 0.176 cm2. The cumulated amount of **1** received as a function of diffusion time was crucial to determine the diffusion properties of **Gn**-**1** complexes. The time of 10% diffused **1** (τ**0.1**) was used as quantitative parameter to compare diffusion efficency. Permeation experiments were repeated 7 times. The mean standard deviation and workup of data were performed as previously (Filipowicz and Wołowiec, 2011).

Fig. 3. The UV-Vis spectrum of **1** in receptor solution.

### **3.2 Results and discussion**

### **3.2.1 Solubilization of 1 in presence of dendrimers studied by the 1 H NMR**

Ascorbic acid (**1**) is well soluble in water, while its solubility in methanol-*d4*, determined by 1H NMR spectroscopy is 0.35 mol⋅dm-3. It increases when bound to PAMAM dendrimers **G2**, **G3**, **G4**, **G2,5** and **G3,5**, respectively. When **1** is dissolved in methanol-*d4*, the doublet resonance of H4 shifts considerably from 4.80 into 4.62 ppm when **1**:**G4** ratio is 1:1. The species formed between **1** and **G3** or **G4** dendrimers become insoluble when the **1**:**Gn** ratio reaches 3:1 and 6:1 for **1**:**G3** and **1**:**G4**, respectively. The isolated complexes are readily water soluble. Presumably their stoichiometry does not correspond to the dendrimers ability to absorb small molecules of ascorbic acid, rather than to the limit of their solubility in methanol.

Opposite to **G3** or **G4** the **G2,5** and **G3,5** dendrimers which have similar sizes and molecular weights act as effective solubilizers of **1** in methanol. They are able to solubilize *ca* 144-160 molecules of **1** per molecule of dendrimer, almost independently on the host size. When methanol is evaporated from such a samples, the separation of **1** from dendrimer is observed resulting in formation of non-homogeneous mixture. In order to establish the stoichiometry of neat host-guest complexes another method was used.

stirred magnetically with 1000 rpm at 32°C. 10 ml aliquots of receptor solution were taken at 0.5 hour or longer time intervals and the receiver compartment was filled with 6 ml portion of a new receptor solution. The experiments were carried on until at least 10% of initial amount of **1** was received in receptor solution. The results were analyzed calculating the flux in [μmol⋅hour-1⋅cm-2]. The active area of membrane determined by size of the ring in Franz cell was 0.176 cm2. The cumulated amount of **1** received as a function of diffusion time was crucial to determine the diffusion properties of **Gn**-**1** complexes. The time of 10% diffused **1** (τ**0.1**) was used as quantitative parameter to compare diffusion efficency. Permeation experiments were repeated 7 times. The mean standard deviation and workup

**200 250 300 350 400 450 500**

Ascorbic acid (**1**) is well soluble in water, while its solubility in methanol-*d4*, determined by 1H NMR spectroscopy is 0.35 mol⋅dm-3. It increases when bound to PAMAM dendrimers **G2**, **G3**, **G4**, **G2,5** and **G3,5**, respectively. When **1** is dissolved in methanol-*d4*, the doublet resonance of H4 shifts considerably from 4.80 into 4.62 ppm when **1**:**G4** ratio is 1:1. The species formed between **1** and **G3** or **G4** dendrimers become insoluble when the **1**:**Gn** ratio reaches 3:1 and 6:1 for **1**:**G3** and **1**:**G4**, respectively. The isolated complexes are readily water soluble. Presumably their stoichiometry does not correspond to the dendrimers ability to absorb small molecules of ascorbic acid, rather than to the limit of their solubility in

Opposite to **G3** or **G4** the **G2,5** and **G3,5** dendrimers which have similar sizes and molecular weights act as effective solubilizers of **1** in methanol. They are able to solubilize *ca* 144-160 molecules of **1** per molecule of dendrimer, almost independently on the host size. When methanol is evaporated from such a samples, the separation of **1** from dendrimer is observed resulting in formation of non-homogeneous mixture. In order to establish the

**3.4 10-5**

 **mol dm-3**

**Spectrum of vitamin C in receiving solution**

**Wavelength [nm]**

**H NMR**

of data were performed as previously (Filipowicz and Wołowiec, 2011).

λ**max = 264nm;** ε**264 = 1.1 x 104**

**0,0**

**3.2 Results and discussion**

methanol.

Fig. 3. The UV-Vis spectrum of **1** in receptor solution.

**3.2.1 Solubilization of 1 in presence of dendrimers studied by the 1**

stoichiometry of neat host-guest complexes another method was used.

**0,1**

**0,2**

**0,3**

**Absorbance**

**0,4**

### **3.2.2 Differential scanning calorimetric studies of neat complexes of 1 with dendrimers**

Recently we have used DSC method to evaluate the stoichiometry of host-guest complexes between PAMAM dendrimers and *8-methoxypsoralene* (Borowska et al., 2010). Water insoluble host formed oily host-guest complexes which revealed higher temperature of glass transition (Tg) than PAMAM dendrimers. When the guest amount was increased above the host maximum capacity, the separation of PAMAM dendrimers and guest took place, followed by Tg returning to its original value. Similar phenomenon was observed in all cases studied here. However, the results on Tg were slightly surprising, because the limit of encapsulation for dendrimers **G3**, **G3,5** and **G4** was 3 molecules of **1** per macromolecule, i.e. lower in comparison with **G2,5**, for which 6 molecules of **1** per molecule of **G2,5**, showed increased value of Tg. However it confirms the 1H NMR results on solubilization in methanol, which also showed that **G2,5** interacts with more molecules of guest than larger hosts. This might be due to different types of interaction between host and **1**. Vitamin C, unlike hydrophobic *8-methoxypsoralene* is able to form ion-pair complex, this interaction prevailing in case of full-generation dendrimers **G3** and **G4**. The ion-pairs decompose when solvent is removed from solution upon preparation of neat samples. Besides, the larger dendrimers are used, the more dense are the cavities of host and self organization of vitamin C crystals prevails over weak intramolecular interaction within host-guest complex. Furthermore we noticed that homogeneous samples of host-guest complexes formed between PAMAM dendrimers and **1** can be obtained in every case when the stoichiometry is maintained as 3:1. These complexes were then used to perform transdermal permeability experiments on skin-model membranes (see below).

Fig. 4. The DSC curves for **1** : **G4** mixtures at molar ratio (from bottom to top): 0:1; 1:1; 2:1; 3:1; 4:1.

### **3.2.3 Permeation studies**

The permeation experiments of **1** through PVDF and PES membranes using **1** dispersed in o/w emulsion indicated a typical change in the flux of **1**; after 0.5 hr induction time, the flux rapidly grew finally stabilizing within 1.5 hour of experiment.

Dermatological Application of PAMAM –

**0,0**

**0**

**2**

**4**

**6**

**8**

**10**

**12 PVDS**

 **PES**

PES membranes in function of PAMAM dendrimer generation.

was 2.8 (± 0.2) mg in every case.

**[hr]** τ**0.1**

**0,1**

**0,2**

**0,3**

**0,4**

**Total amount of 1 [mg]**

**0,5**

Vitamin Bioconjugates and Host-Guest Complexes – Vitamin C Case Study 205

useful for controlling transdermal diffusion of dermatologically important agents. Finally the emulsion containing vitamin and dendrimeic carrier can be easily prepared by addition of pre-formed host-guest complex in ethanol and addition of oily complex to the prepared emulsion. Such prepared creams may carry both water-soluble and water-insoluble vitamins, as well as some vitamin derivatives bonded covalently, like retinal, pyridoxal or

**0 1 2 3 4 5 6 7 8 9 10 11**

**01234**

Fig. 6. The dependence of τ**0.1** in experiments of transdermal diffusion through PVDS and

**Generation**

Fig. 5b. The cumulated amount of **1** diffused through PES membrane vs time. The load of **1**

**Time [hr]**

biotin. These bioconjugates are currently under investigation in our laboratory.

**0,6 NONE**; mean SD = 0.012  **G2**; mean SD = 0.011  **G2.5**; mean SD = 0.004  **G3**; mean SD = 0.003  **G3.5**; mean SD = 0.003  **G4**; mean SD = 0.010

Addition of PAMAM dendrimers of **G2**, **G2,5**, **G3**, **G3,5**, and **G4** only slightly influenced the rate of diffusion through PVDS membrane. The experiments were conducted until ca 50% of **1** was transferred through PVDS and the time of 10% transfer, τ**0.1**. was used as reference in all the experiments. Addition of smaller size dendrimers **G2**, **G2,5**, **G3** caused elongation of τ**0.1**, presumably due to host-guest complexes diffusion or diffusion of **1** preliminarily released from them in emulsion. The larger size dendrimers **G3,5**, and **G4** had no such impact (Figure 5a). Generally the diffusion of **1** through PVDF was fast, with 10% of **1** transferred within ca 1 hour. Unlikely, the diffusion through much thicker PES membrane occurred slower being strongly affected by the added dendrimer (Figure 5b). This influence could be clearly demonstrated by comparison of τ**0.1** (Figure 6 and Table 2). The slowest diffusion was found in case of **1**-**G2,5** composition. The diffusion rate was diminished by a factor of 4 if compared with **1** itself. **G3** considerably decreased the rate by a factor of 3.7, while other dendrimers, larger **G3,5** and **G4** and smaller **G2**, showed lower impact. The results obtained for diffusion experiments through PES confirm the PVDF ones, nevetheless remarkable differentiation of diffusion efficiency was found in case of PES.

Fig. 5a. The cumulated amount of **1** diffused through PVDF membrane vs time. The load of **1** was 2.8 (± 0.2) mg in every case.

Comparing the supposed stoichiometry of host-guest complexes based on DSC measurements as well as remarkable solubilization of **1** in methanol containing **G2,5** and **G3,5** we can assume that these dendrimers form the most stable host-guest complexes, with **1** encapsulated within macromolecule, while **G3** and **G4** encapsulate **1** weakly to give the 3:1 **1**: **Gn** complexes. Moreover, **1** interacts with **G2**, **G3** and **G4** surface amine groups with this interaction prevailing on encapsulation. When **1** is released from ionic complexes with **Gn**, it diffuses similarly like from emulsion. When the whole host-guest complex diffuse, the rate is considerably lower. This phenomenon is quite opposite to what was observed for the systems in which PAMAM dendrimers were used as solubilizers of water-insoluble *psoralene* or *riboflavin* (Borowska et al, 2010, Filipowicz and Wołowiec, 2011); in these cases the transdermal diffusion of these compounds was enhanced. Both of these behaviors are

Addition of PAMAM dendrimers of **G2**, **G2,5**, **G3**, **G3,5**, and **G4** only slightly influenced the rate of diffusion through PVDS membrane. The experiments were conducted until ca 50% of **1** was transferred through PVDS and the time of 10% transfer, τ**0.1**. was used as reference in all the experiments. Addition of smaller size dendrimers **G2**, **G2,5**, **G3** caused elongation of τ**0.1**, presumably due to host-guest complexes diffusion or diffusion of **1** preliminarily released from them in emulsion. The larger size dendrimers **G3,5**, and **G4** had no such impact (Figure 5a). Generally the diffusion of **1** through PVDF was fast, with 10% of **1** transferred within ca 1 hour. Unlikely, the diffusion through much thicker PES membrane occurred slower being strongly affected by the added dendrimer (Figure 5b). This influence could be clearly demonstrated by comparison of τ**0.1** (Figure 6 and Table 2). The slowest diffusion was found in case of **1**-**G2,5** composition. The diffusion rate was diminished by a factor of 4 if compared with **1** itself. **G3** considerably decreased the rate by a factor of 3.7, while other dendrimers, larger **G3,5** and **G4** and smaller **G2**, showed lower impact. The results obtained for diffusion experiments through PES confirm the PVDF ones, nevetheless

**01234567**

**Time [hr]**

Fig. 5a. The cumulated amount of **1** diffused through PVDF membrane vs time. The load of

Comparing the supposed stoichiometry of host-guest complexes based on DSC measurements as well as remarkable solubilization of **1** in methanol containing **G2,5** and **G3,5** we can assume that these dendrimers form the most stable host-guest complexes, with **1** encapsulated within macromolecule, while **G3** and **G4** encapsulate **1** weakly to give the 3:1 **1**: **Gn** complexes. Moreover, **1** interacts with **G2**, **G3** and **G4** surface amine groups with this interaction prevailing on encapsulation. When **1** is released from ionic complexes with **Gn**, it diffuses similarly like from emulsion. When the whole host-guest complex diffuse, the rate is considerably lower. This phenomenon is quite opposite to what was observed for the systems in which PAMAM dendrimers were used as solubilizers of water-insoluble *psoralene* or *riboflavin* (Borowska et al, 2010, Filipowicz and Wołowiec, 2011); in these cases the transdermal diffusion of these compounds was enhanced. Both of these behaviors are

 **None**; mean SD = 0.015  **G2**; mean SD = 0.016  **G3**; mean SD = 0.015  **G2.5**; mean SD = 0.014  **G3.5**; mean SD = 0.017  **G4**; mean SD = 0.014

remarkable differentiation of diffusion efficiency was found in case of PES.

**0,2**

**1** was 2.8 (± 0.2) mg in every case.

**0,3**

**0,4**

**Total amount of 1 [mg]**

**0,5**

**0,6**

**0,7**

**0,8**

useful for controlling transdermal diffusion of dermatologically important agents. Finally the emulsion containing vitamin and dendrimeic carrier can be easily prepared by addition of pre-formed host-guest complex in ethanol and addition of oily complex to the prepared emulsion. Such prepared creams may carry both water-soluble and water-insoluble vitamins, as well as some vitamin derivatives bonded covalently, like retinal, pyridoxal or biotin. These bioconjugates are currently under investigation in our laboratory.

Fig. 5b. The cumulated amount of **1** diffused through PES membrane vs time. The load of **1** was 2.8 (± 0.2) mg in every case.

Fig. 6. The dependence of τ**0.1** in experiments of transdermal diffusion through PVDS and PES membranes in function of PAMAM dendrimer generation.

Dermatological Application of PAMAM –

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Table 2. The time of 10% transfer of **1** (τ**0.1**) from emulsions containing dendrimers.

### **4. Conclusions**

### G2,5≅G3 > G3,5 >> G2≅G4 >none

5. Finally, the **G2,5** and **G3** dendrimers can be applied as transdermal carriers to control vitamin C release kinetics from emulsions or hydrogels. Vitamin C retardation is surprisingly in contrast with previously studied water insoluble 8-methoxypsoralene or riboflavin which showed a promotion of the diffusion process.

### **5. Acknowledgment**

The work was supported by the Grant no N N302 432839, obtained from Ministry of Higher Education and Research, Poland.

### **6. References**

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[hr]

Table 2. The time of 10% transfer of **1** (τ**0.1**) from emulsions containing dendrimers.

1. PAMAM dendrimers influence the solubility of ascorbic acid (**1**) in methanol. The solubility of ionic complexes between **1** and full-generation PAMAM dendrimers in methanol is lower in comparison with **1** itself. 3:1 stoichiometry adducts between vitamin **1** : **G3** and **1** : **G4**, can be isolated from methanolic solutions as homogeneous,

2. Half-generation PAMAM dendrimers: **G2,5** and **G3,5** solubilize **1** in methanol due to non-specific interaction up to *ca* 150 molecules of **1** per molecule of dendrimer. 3. The ionic interactions, encapsultation, and surface absorption of **1** by PAMAM dendrimers result in host-guest complexes formation. Based upon DSC measurements the stoiochiometry of the homogeneous, oily complexes was determined as 6:1 (for **1** :

4. When 10% of **1** per dendrimer compositions are placed in o/w emulsions, the permeation profile of **1** through the polyvinyldifluoride (PVDF) and pig ear skin (PES) membranes depends on the presence and size of dendrimer used as vitamin carrier in the emulsion. Transdermal diffusion through PES is considerably slowed down by

G2,5≅G3 > G3,5 >> G2≅G4 >none 5. Finally, the **G2,5** and **G3** dendrimers can be applied as transdermal carriers to control vitamin C release kinetics from emulsions or hydrogels. Vitamin C retardation is surprisingly in contrast with previously studied water insoluble 8-methoxypsoralene or

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**1** 0.9 (±0.12) 3.0 (±0.21) **1-G2** 1.3 (±0.15) 4.5 (±0.26) **1-G2.5** 1.3 (±0.15) 12.0 (±0.29) **1-G3** 1.0 (±0.13) 11.0 (±0.28) **1-G3.5** 0.9 (±0.12) 8.2 (±0.20) **1-G4** 0.9 (±0.12) 3.7 (±0.22)

τ**0.1**

PVDS PES

Sample (o/w)

**4. Conclusions**

solvent-free oily complexes.

**G2,5**) and 3:1 (for **1** : **G3**, **1** : **G3,5** and **1** : **G4**).

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**Part 4**

**The Role of Stoichiometry**

**in the Determination of Protein Interactions**


**1. Introduction**

Clinically, interaction between organs is of growing relevance given the increasing number of elderly patients with many comorbidities and the recognition that such comorbidities not only influence the clinical course of a given disease and its prognosis, but also affect treatment options and therapeutic success [1]. To exemplify, impaired kidney function associates with poor outcome mainly due to a high burden of cardiovascular (CV) comorbidity, with its manifestations of ischemic heart disease, heart failure, or CV death—a major public health burden in developed countries [2]. Patients with chronic kidney disease (CKD) exhibit a more than four-fold (CKD stage > 3) higher CV risk compared to the non-CKD cohort [2]. Traditional strategies to reduce this risk are largely ineffective in CKD and end-stage renal disease (ESRD) patients, underscoring the importance of non-traditional CKD-specific CV risk factors that are hitherto unknown [2]. In the general population, classical atherosclerotic endpoints such as stroke or myocardial infarction are the dominant cause of death. Importantly, CKD

patients mostly die from sudden cardiac death or ischemic heart disease due to premature vascular and cardiac aging [2].

CKD impairs the removal of harmful substances from the body. Therefore dialysis therapy is required to supplant the most important functions of the kidney. During dialysis, waste products like uremic toxins and excess salts and liquids are discharged via di ffusion through a semipermeable dialysis membrane. Because of the small pore size of currently used dialysis membranes, protein-bound uremic toxins cannot be filtered during dialysis [3,4], leading to their presence in the blood of CKD patients, which might play a role in the development of cardiovascular disease (CVD). The protein-bound uremic toxin indoxyl sulfate is associated with CV death, and its levels correlate positively with CV calcification [5]. Thus, the existent dialysis therapy remains insu fficient, which may explain the poor prognosis of ESRD patients [3].

These patients su ffer from abnormalities in mineral metabolism, caused by an imbalance of calcification promoters (e.g., calcium and phosphate) and inhibitors (e.g., matrix Gla protein (MGP) and fetuin-A) [6] and termed 'mineral bone disorder' (MBD). The interconnection of phosphate, calcium, 1,25-dihydroxycholecalciferol (1,25(OH)2D), and fibroblast growth factor 23 (FGF-23) a ffects the kidney–parathyroid gland–bone axis [7]. In early CKD stages, physiological phosphate serum levels can be sustained. Renal phosphate is restricted by a decreasing glomerular filtration rate (GFR), causing hyperphosphatemia—a major challenge in CKD–MBD. In response to high serum phosphate, osteoblasts produce FGF-23, which inhibits 1,25(OH)2D production. Deficiency in 1,25(OH)2D lowers the serum calcium levels that stimulate the parathyroid gland to produce parathyroid hormone (PTH). The secondary hyperparathyroidism (sHPT) induces calcium e fflux from the bone, leading to low bone mineral density. Vitamin D analogs and calcimimetics are used to suppress PTH. Bisphosphonates inhibit osteoclast activity and are applied to treat the dysregulated bone metabolism in CKD–MBD (Figure 1) [8]. Alterations of the bone mineral density are associated with the progression of aortic calcification in women but not in men [9]. Especially, postmenopausal women exhibit an increased risk for CV events [10]. The International Society of Nephrology (ISN) recommends frequent monitoring of serum levels of calcium, phosphate, and PTH, starting in CKD stage 3 patients [11]. If necessary, patients should be treated to maintain an age-appropriate physiological range of serum parameters.

**Figure 1.** Pathogenesis of chronic kidney disease–mineral bone disorder (CKD–MBD). Targets for therapeutic strategies are written in red; 1,25(OH)2D: 1,25-dihydroxycholecalciferol (calcitriol). The figure was partially created using Servier Medical Art, licensed under a Creative Commons Attribution 3.0 Unported License. Black arrows indicate an increase.

Modifications in the circulation as well as in the myocardium are crucially involved in the increased CV risk in CKD patients. However, both the mediators and the underlying molecular mechanisms remain largely unexplored [12]. CV calcification—both in the tunica intima and in the media—is massively increased in CKD patients and is an independent risk factor for CV morbidity and mortality [13]. CV calcification could be one of the key mechanisms leading to increased CVD in CKD. CV calcification results from active cellular processes in which smooth muscle cells undergo phenotypic changes to build a mineralized matrix [14]. This process is supported by an imbalance of promoters and inhibitors of calcification, which promotes calcium and phosphate precipitation [15,16]. CKD patients with CV risk suffer from mineral deposits in the tunica media (arteriosclerosis) and tunica intima (atherosclerosis). The extent of CV calcification depends on the CKD stage [16,17]. Both traditional and non-traditional risk factors of CV calcification lead to the manifestation of CVD in CKD (Figure 2) [18,19].

**Figure 2.** Traditional and non-traditional CVD risk factors affect uremia-induced calcification. Calcification in CKD can result within the tunica intima and tunica media. CVD, cardiovascular disease; The figure was partially created using Servier Medical Art, licensed under a Creative Commons Attribution 3.0 Unported License. Arrows indicate risk factors, which are present in CKD patients suffering from CVD.

Current treatments induce no adequate reduction of CV calcification in CKD, rendering the identification and development of promising therapeutic targets essential. Experimental rodent CKD models proffer novel promising treatments; for example, the hexasodium salt of myo-inositol hexaphosphate SNF472 has been suggested as a potent ectopic calcification inhibitor both in vitro and in vivo [20,21]. Recent studies suggest the peroxisome proliferator-activated receptor-gamma (PPARγ) and the mineralocorticoid receptor (MR) as novel molecular targets for CV complications in CKD. This review focuses on new potential treatments and compares their benefits in non-transgenic animal models with those in human clinical trials and meta-analyses.


**Why should one normalize the expression data?**

RNASeq experiments have multiple sources of systematic variations introduced through intersample differences such as difference in library size (sequencing depth) or unwanted variations due to batch effects such as sampling time or different sequencing technology [12] or through intra-sample differences such as difference in read length [88] or GC content between genes [89, 90]. These variations, if ignored, can dramatically reduce the accuracy of statistical inference and hence should be removed or controlled during statistical analysis. Therefore, read count and FPKM of a feature, as calculated for example by htseq-count and Cufflinks, respectively, may not be appropriate to compare across features and samples without nor‐ malization.

Normalization is a process that aims to ensure that expression estimates are comparable. There are a number of normalization methods, such as:

**f.** Median of ratio: the normalization factor for each sample is computed as the median of ratios of expressions of features over their geometric means across all samples. Finally, each feature expression is divided by this factor to get the normalized expression [96]. Median of ratio has been implemented in R packages DESeq [96], DESeq2 [97], and in Cuffdiff2 [92].

Several publications [98, 99] comparing normalization methods suggest that median of ratio is the best method for normalization in differential expression study for mRNASeq experiment.

In addition to normalization methods, several packages have been developed to control batch effects, for example, svaseq [100]. svaseq can work on both, count-based data (e.g., htseq-count generated data) as well as FPKMs (Cufflinks generated data).


**About the Editors**

**Ryszard Strzelecki** (M'97-SM'07) was born in Bydgoszcz, Poland. He received the M.Sc. and Ph.D. degrees in Electronic Engineering from the Department of Industrial Electronics, National Technical University of Ukraine "Kyiv Polytechnic Institute", Kyiv, Ukraine, in 1981 and 1984, respectively, and the Dr. Sc. degree in Electrical Engineering from the Institute of Electrodynamics, The National Academy of Sciences of Ukraine, Kyiv, in 1991. He is currently a full professor with the Gdansk University of Technology (Gda ´ nsk, Poland), co-head of the Laboratory of Power ´ Electronics and Automated Electric Drive, ITMO University (St. Petersburg, Russia), and Scientific Consultant for Power Electronics to the Management Board of AREX Ltd. In 2020, he was elected to the Committee on Electrical Engineering of the Polish Academy of Sciences. His research activity is concentrated on the topology, control, and industry application of power electronic conditioners, particularly for power quality enhancement and power flow control on distributed electrical networks.

**Galina Demidova** received her B.Sc., engineer, and Ph.D. degrees from ITMO University, Saint Petersburg, Russia. At present she is an Associate Professor at the Faculty of Control Systems and Robotics, Engineer of the Industrial Department of Precision Electromechanical Systems, and researcher of the International Scientific Laboratory "Power electronics and Automated Electric Drive ", at ITMO University. She has performed more than 20 R&D projects, dealing with control systems in digital electric power drives for tracking telescopes. She has authored or coauthored more than 50 conference and journal papers. She is co-Guest Editor in several Special Issues, and an organizer of the IEEE Conference. Her current research interests include adaptive control, fuzzy logic control, motion control, automatic control, fuzzy neural networks, hybrid intelligent systems, genetic algorithms, electromechanical systems, wind turbines, and multi-agent control

**Dmitri Vinnikov** Guest Editor 3: Prof. Dr. Dmitri Vinnikov Dmitri Vinnikov was born in Tallinn, Estonia, in 1976. He received the Dipl.Eng., M.Sc., and Dr.Sc.techn. degrees in Electrical Engineering from Tallinn University of Technology, Tallinn, Estonia, in 1999, 2001, and 2005, respectively. He is currently a Research Professor and the Head of the Power Electronics Group, Department of Electrical Power Engineering and Mechatronics, Tallinn University of Technology (Estonia), and a visiting professor in the Faculty of Control System and Robotics, ITMO University, Saint Petersburg, Russia. He is the Head of R&D and co-founder of Ubik Solutions LLC.— an Estonian start-up company dedicated to innovative & smart power electronics for renewable energy systems. Moreover, he is one of the founders and leading researchers of ZEBE—the Estonian Centre of Excellence, performing research toward the development of zero energy and resource efficient smart buildings and districts. He has authored or co-authored two books, five monographs, and one book chapter, as well as more than 300 published papers on power converter design and development, and is the holder of numerous patents and utility models in this field. His research interests include applied designs of power electronic converters and control systems, renewable energy conversion systems (photovoltaic and wind), impedance-source power converters, and implementation of wide bandgap power semiconductors. D. Vinnikov is a Chair of the IEEE Estonia Section .


**Meet the editor**

Heimo Mikkola received his PhD degree in Applied Zoology and Limnology from the University of Kuopio, Finland. Since 1988, he has been an adjunct Professor of the Eastern Finland University through which he has lately been a part-time visiting professor in the Kazakh National Agrarian University in Almaty lecturing on fisheries and aquaculture to Master's level students and

on Green Biotechnology and Global Food Security to all university teachers. Before and in between his university career, he has worked abroad for well over 30 years in Africa, South America and Central and Southeast Asia mainly with the African Development Bank and Food and Agriculture Organization of the United Nations (FAO). During these years, he had an excellent free time opportunity to visit and study owls in over 130 countries of the world. Owl studies often took him to the best bat biotopes as well, and he started to collect data on bats eaten by owls. In 2014, he was given the title "Champion of Owls" in Houston, USA, mainly because of his six worldwide distributed and translated owl books.

Contents

**Preface VII**

Heimo Mikkola

**in Algeria 21** Mourad Ahmim

Vasil V. Popov

**Pakistan 81** Mohammad Salim

Nicholas Johnson

**Monitoring of Bats 7**

**Distribution Models 39**

**Temperate Forests 63**

Chapter 1 **Introductory Chapter: Bats Eaten by Owls 1**

Chapter 2 **Comparison of Driving Transect Methods for Acoustic**

Chapter 4 **Bats in Bulgaria: Patterns of Species Distribution, Richness,**

Aaron S. Hogue and Andrew T. McGowan

Chapter 3 **The Bat: A Benefactor Animal Poorly Understood**

**Rarity, and Vulnerability Derived from**

Chapter 5 **Bat Conservation Management in Exploited European**

Laurent Tillon, Joseph Langridge and Stéphane Aulagnier

Chapter 6 **Bats in Northern Mountain Region of Khyber Pakhtunkhwa,**

Chapter 7 **The Daubenton's Bat (Myotis daubentonii, Kuhl, 1817) and Its**

Chapter 8 **Bats, Bat-Borne Viruses, and Environmental Changes 113**

**Role as a Reservoir for Europe Bat Lyssavirus Type-2 101**

Aneta Afelt, Christian Devaux, Jordi Serra-Cobo and Roger Frutos