International Journal of Biological Macromolecules 163 (2020) 96–107

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Synthesis, antimicrobial activity, and sustainable release of novel

α-aminophosphonate derivatives loaded carrageenan cryogel
Dalia A. Elsherbiny a,b, Abdelrahman M. Abdelgawad b,c, Mehrez E. El-Naggar c,⁎, Ramy A. El-Sherbiny a,
Mohamed H. El-Rafie c, Ibrahim El-Tantawy El-Sayed a,⁎
a
Menoufia University, Faculty of Science, Chemistry Department, Shebin El-Kom, Menoufia, Egypt
b
Aachen-Maastricht Institute for Biobased Materials, Faculty of Science and Engineering, Maastricht University, the Netherlands
c
Textile Research Division, National Research Center (Affiliation ID: 60014618), Dokki, Cairo, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Novel α-aminophosphonates 4 were synthesized via one pot three-component reaction of 4-aminoantipyrine,
Received 22 May 2020 aldehydes, triphenylphosphite and Lewis acid catalyst. The chemical structures of all the synthesized compounds
Received in revised form 6 June 2020 were elucidated by IR, NMR and MS spectral analysis. The antimicrobial activity of 4 was tested in vitro against
Accepted 25 June 2020
pathogenic microbes such as E. coli, S. aureus, A. niger and C. albicans. Three of them (4f–h) exhibited high anti-
Available online 29 June 2020
microbial activity and were loaded to carrageenan cryogel for drug delivery studies. With the aid of cellulose
Keywords:
nanofibrils (CNF) as reinforcing material and glyoxal as a cross-linking agent, porous cryogels with improved me-
Carrageenan chanical properties were obtained. Among all, CAR-7 presents the optimum cryogel sample, which contained
Cryogel around 16% CNF and 0.2 mL/15 mL polymer blend. CAR-7 demonstrated highest mechanical compressive
Antimicrobial activity and sustainable release strength, porosity (80%), and swelling capacity (75%). Sustainable release behavior over 24 h was observed for
the loaded cryogels. The antimicrobial activity of cryogels against S. aureus showed marginal differences between
samples. CAR-9 (loaded with 4f) showed the highest reduction percentage in number of bacterial colonies
(99.94%) followed by CAR-11 (loaded with 4h, 99.3%) and finally CAR-10 (loaded with 4g, 99.29%).
© 2020 Elsevier B.V. All rights reserved.

1. Introduction activities [16,17]. For the current piece of research, we are revealing
the preparation of ten novel α-aminophosphonate subordinates based
The accumulative microbial resistance to current antibiotics forces on 4-aminoantipyrene utilizing an efficient one pot and three-
the development of active molecules with possible activity against path- component synthesis strategy.
ogenic microorganisms [1]. α-Aminophosphonic acid and their corre- One of the significant concerns in regards to the versatile uses of the
sponding derivatives were thoroughly studied in recent years due to heterocyclic compounds is their low remedial impacts because of poor
their wide range of environmental [2–4], and biological applications as fluid dissolvability and absence of similarity with hydrophilic delivery
herbicides [5], antibacterial [6–8], antiviral [9], antifungal [10], and anti- frameworks [18]. This restriction can be defeated in any event to some
cancer agents [11–13]. This class of organophosphorus compounds pos- degree by planning the best possible delivery system. Several drug de-
sesses high biological efficacy, metabolic stability and insignificant livery vehicles have been investigated in the literature such as emul-
toxicity towards mammalian cells. Therefore, α-aminophosphonate sions, microspheres, nanoparticles, hydrogels and aerogels. Among
moiety was recognized a unique pharmacophore from drug design per- those systems; aerogels, cryogels, and xerogels attracted much atten-
spective [14,15]. Of late, different approaches have been explored for tion due to their porous structure as well as large surface area
the synthesis of aminophosphonates to improve its achievability. The (400–1200 m2 g−1) [19]. The inherited nano- and micro-pores makes
synthetic pathways of α-aminophosphonates depend on the nature of such materials decent drug carrier candidates [20,21]. Aerogels,
reactants and catalyst. It is qualified to specify that the most outstanding cryogels, and xerogels hold different levels of porosity and can be pre-
advances in therapeutic science have been made when N-heterocyclic pared using different techniques. The most porous, aerogel, exhibit
mixes were coordinated in the atomic structure of the drugs. Among very high surface area and is fabricated via supercritical carbon dioxide
them all, antipyrine and its analogs display a wide scope of biological drying. On contrary, xerogels have relatively rigid structure with less
noticeable pores and consequently, lower surface area. Cryogels possess
⁎ Corresponding authors.
intermediate properties between aerogels and xerogels and fabricated
E-mail addresses: [email protected] (M.E. El-Naggar), via freeze-drying technique [22]. Hydrophobic drugs as well as insolu-
[email protected] (I.E.-T. El-Sayed). ble functional materials (metal nanoparticles, nanocellulose and carbon

https://doi.org/10.1016/j.ijbiomac.2020.06.251
0141-8130/© 2020 Elsevier B.V. All rights reserved.
 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107 97

nanotubes) can be dispersed in appropriate media and incorporated in acetonitrile lithium perchlorate (10 mol%) was added portionwise
these gel systems [23]. Cryogels from several polysaccharides (cellulose, under stirring at room temperature. The reaction mixture was further
gelatin, and curdlan) were reported as efficient drug carriers [24–27]. stirred at room temperature and the progress of the reaction was mon-
Carrageenan, which is a linear sulfated polysaccharide was recently itored by the thin layer chromatography (TLC) till all the starting mate-
recognized as a drug delivery vehicle/or system [28,29]. It is generally rials were consumed, after 5 days. The precipitated product was filtered
separated from red seaweeds and regularly utilized in the food industry, off followed by crystallization from methanol and cooling to afford pure
in view of its gelling and thickening properties [30–34]. Carrageenan 4 in good yield.
inherited anionic behavior due to the presence of the chemically active
ester-like sulfates. In its wet state, carrageenan can form very strong 2.3.1.1. Diphenyl ((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-
three-dimensional gels, however, its dry structure possess poor me- 4-yl)amino)(4 (dimethylamino)phenyl)methyl)phosphonate (4a). Yield:
chanical strength so that the incorporation of enforcing agents is favor- 60%, IR (KBr) cm−1: 3681 (NH), 1642 (C_O), 1580 (C_C, Ar.), 1293
able [19]. (P_O), 1228 (N\\C), 765 (P\\C). 1HNMR (DMSO‑d6 500 MHz):
Herein, we present the synthesis of ten novel α-aminophosphonate −2.39 (s, 3H, CH3), 2.49 (s, 6H, 2CH3), 2.96–3.09 (m, 4H, CH),
derivatives by the use of 4-aminoantipyrine and various aldehydes. 6.73–7.62 (m, 19H, CHAr), 9.42 (s, 1H, NH). 13CNMR (DMSO‑d6
Triphenylphosphite was incorporated as a source of phosphorus nu- 500 MHz): −111.676, 117.609, 124.048, 125.259, 126.489, 128.750,
cleus and lithium perchlorate as a catalyst. The synthesized compounds 129.084, 134.864, 151.356, 151.747, 155.410, 160.150. LCMS, m/z
were fully characterized in order to prove its chemical structures. The (C32H33N4O4P) calcd, 568.61; found, 569.15 [M + 1]+.
biological activity of the prepared compounds was qualitatively
screened using the disc diffusion method against Gram-negative bacte- 2.3.1.2. Diphenyl ((4-bromophenyl)((1,5-dimethyl-3-oxo-2-phenyl-2,3-
ria, E. coli; Gram-positive bacteria, S. aureus; a fungus, A. niger; and a dihydro-1H-pyrazol-4-yl)amino)methyl)phosphonates (4b). Yield
yeast, C. albicans. Compounds with highest antimicrobial efficacy, 3 can- (78.5%), IR (KBr) cm−1: −3661 (NH), 1643 (C_O), 1582 (C_C, Ar),
didates, were selected to be loaded to carrageenan cryogel system with 1297 (P_O), 758 (P\\C), 626 (C\\Br). 1HNMR (DMSO‑d6 500 MHz):
a hope to introduce appropriate delivery system for wound care appli- −2.48 (s, 3H, CH), 3.18 (s, 3H, NH), 3.33 (s, 1H, CH), 7.35–7.76 (m,
cations. The antimicrobial activity of these candidates was further inves- 19H, CHAr), 9.53 (s, 1H, NH). 13CNMR (DMSO‑d6 500 MHz): −9.740,
tigated against S.aureus, and E. coli quantitatively via cell viable counting 115.950, 123.361, 124.763, 127.043, 129.017, 129.179, 131.745,
method. Carrageenan/cellulose nanofibrils (CAR/CNF) cryogel system 134.444, 136.743, 152.233, 152.701, 159.435. LCMS, m/z
was fabricated via freeze-drying technique and used as a hydrophilic (C30H27BrN3O4P) calcd, 604.44; found, 605.05 [M + 1]+.
bio-based drug carrier. Cellulose nanofibrils (CNF) is being used in the
current cryogel system to improve the three-dimensional stability. 2.3.1.3. Diphenyl(((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
CNF was first produced with lateral dimensions in nanometer range pyrazol-4-yl)amino)(perfluorophenyl)methyl)phosphonate(4c). Yield
by passing the cellulose pulp through high pressure homogenizer [35] (75%), IR (KBr) cm−1: −3682 (NH), 1647 (C_O), 1572 (C_C, Ar.),
who such type of treatment results in strongly entangled nanofibrils 1375 (C\\F), 1297 (P_O), 762 (P\\C). 1HNMR (DMSO‑d6 500 MHz):
network. Normally, CNF have a range of 5–50 nm in diameter and a sev- −2.99 (s, 3H, CH3), 3.00 (s, 3H, NH), 3.38 (s, 1H, CH), 7.352–7.553 (m,
eral micrometers in length [36]. Up apparently, this cryogel framework 15H, CHAr), 9.608 (s, 1H, NH)., 13CNMR (DMSO‑d6 500 MHz): 9.559,
has not been examined in the literature previously. 115.530, 125.478, 127.586, 129.170, 129.275, 133.977, 141.484,
152.214, 158.672. LCMS, m/z (C30H23F5N3O4P) calcd, 615.50; found,
2. Experimental 616.30 [M + 1]+.

2.1. Materials 2.3.1.4. Diphenyl ((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-

4-yl)amino)(4-hydroxy-3-methoxyphenyl)methyl)phosphonates (4d).
Carrageenan (CAR, Product code: P10011037568) and β- Yield (65%), IR (KBr)cm−1: 3430 (NH), 1595 (C_O), 1495 (C_C, Ar.),
cyclodextrin were purchased from Sigma-Aldrich Co. (USA). Wood fi- 1261 (P_O), 767 (P\\C). 1HNMR (DMSO‑d6 400 MHz): 2.428 (s, 3H,
bers were submitted to mechanical dismantling with microfluidizerto CH3), 3.116 (s, 3H, NH), 3.840 (s, 3H, CH), 6.847–6.874 (m, 4H, CHAr),
produce cellulose nanofibrils (CNF). Acetonitrile and glyoxal were pur- 7.187–7.540 (m, 15H, CHAr), 9.475 (s, 2H). 13CNMR (DMSO‑d6
chased from Panreac qumicns A Co. (India). The materials and solvents 500 MHz): 9.826, 55.530, 109.609, 115.467, 117.057, 122.115,
required for synthesis of α-aminophosphonates such as acetonitrile 124.270, 126.645, 129.092, 129.350, 134.772, 148.003, 149.202,
were purchased and used without further purification. For the prepara- 151.656, 155.086, 159.962.
tion of solutions and suspensions, deionized water has been used.
2.3.1.5. Diphenyl ((4-cyanophenyl)((1,5-dimethyl-3-oxo-2-phenyl-2,3-
2.2. Characterization of materials dihydro-1H-pyrazol-4-yl)amino)methyl)phosphonate (4e). Yield (70%),
IR (KBr) cm−1: −3681 (NH), 2218 (C`N), 1642 (C_O), 1558 (C_C,
All 1H NMR and 13C NMR testing (solvent DMSO‑d6) were performed Ar.), 1206 (P_O), 763 (P\\C). 1HNMR (DMSO‑d6 400 MHz): 2.90 (s,
at Mansoura University using 500 MHz varian, 500 MHz varian and 3H, CH), 3.47 (s, 3H, NH), 6.20 (s, 1H, CH), 6.93–6.96 (t, J = 7.5, 2H,
Bruker Avance. Thermo Nicolet (USA) was used for FTIR spectroscopy. CHAr), 7.03–7.05 (d, 4H, CHAr), 7.19–7.40 (m, 9H, CHAr), 7.51–7.54 (t,
Chemical changes that occur related to the respective solvent were re- J = 8.5, 2H, CHAr), 7.87–7.98 (dd, j = 34.5, 2H, CHAr), 9.59 (s, 1H, NH).
13
corded in ppm. On Shimadzu QL 800 15–70 V, at the Faculty of Science CNMR (DMSO‑d6 500 MHz):- 120.356, 120.394, 121.797, 125.135,
and Engineering of the Netherlands' Maastricht University, mass spec- 127.625, 128.969, 129.237, 132.699, 153.426 LCMS, m/z
troscopy experiments were recorded. The reactions were controlled (C31H27N4O4P) calcd, 550.55; found, 551.50 [M + 1]+.
and monitored via TLC (Merck) on Kiesel gel F254 precoated plates.
2.3.1.6. Diphenyl ((3-chlorophenyl)((1,5-dimethyl-3-oxo-2-phenyl-2,3-
2.3. Methods dihydro-1H-pyrazol-4-yl)amino)methyl)phosphonate (4f). Yield (75%),
IR (KBr) cm−1: 3681(NH), 1640 (C_O), 1592 (C_C, Ar.), 1284
2.3.1. General procedure for the Synthesis of α-aminophosphonate (P_O), 766 (P\\C), 740 (C\\Cl). 1HNMR (DMSO‑d6, 500 MHz): 2.46
derivatives 4 (s, 3H, CH3), 3.39 (s, 3H, NH), 6.20 (s, 1H, CH), 6.94–6.97 (t, J = 7.5,
To a mixture containing equimolar amounts of aldehydes 1, 4- 2H, CHAr), 7.047–7.063 (d, 4H, CHAr), 7.19–7.40 (m, 9H, CHAr),
aminoantipyrine 2, and triphenylphosphite 3 dissolved in 10 mL of 7.355–7.542 (m, 4H, CHAr), 9.536 (s, 1H, NH). 13CNMR (DMSO‑d6
98 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107

500 MHz): −9.769, 120.366, 120.414, 121.892, 124.868, 125.927, could be achieved. The plates were then incubated with bacteria at
126.289, 127.119, 128.998, 129.208, 129.704, 130.696, 152.186, 37 °C throughout 24 h and put vertical for 48 h at 30 °C in order to facil-
152.329, 153.321, 153.378. LCMS, m/z (C30H27ClN3O4P) calcd, 559.99; itate the maximal organism growth. By calculating the diameter of the
found, 561.30 [M + 1]+. inhibition zone expressed in millimeter (mm), the antimicrobial activity
of the tested samples against microbe species was recorded.
2.3.1.7. Diphenyl ((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-
4-yl)amino)(2-hydroxyphenyl)methyl)phosphonates (4g). Yield (72%), IR 2.3.3. Determination of antimicrobial efficacy of selected drugs via colony
(KBr) cm−1: 3681 (NH), 1593 (C_O), 1686 (C_C, Ar.), 1284 (P_O), forming unit (CFU) method
748 (P\\C). 1HNMR (DMSO‑d6 400 MHz): 2.403 (s, 3H, CH3), 2.763 (s, S. aureus and E. coli bacteria (100 μl stock of 7.4 × 107 CFU) were in-
3H, NH), 3.204 (s, 1H, CH), 7.352–7.553 (m, 19H, CHAr), 9.706 (s, 2H). oculated in a 10 mL of liquid nutrient for 24 h (pH 6.8) that was freshly
13
CNMR (DMSO‑d6 500 MHz): 9.738, 114.067, 115.725, 119.095, prepared; 100 mL contains peptone (5 g/L), beef extract (3 g/L). In the
124.236, 125.890, 127.806, 128.588, 129.183, 129.256, 131.008, inoculated flasks (with 20 μL of inoculums), samples have been inserted
131.60, 150.02, 157.556, 159.408. LCMS, m/z (C30H28N3O5P) calcd, leaving the control (sample-less inoculated flasks). A serial dilution of
541.54; found, 542.30 [M + 1]+. each cultivation containing a sample and control (10−1–10−9) was car-
ried out after 16 h of incubatation at 37 °C. The colony-forming units
2.3.1.8. Diphenyl((3,4-dihydroxyphenyl)((1,5-dimethyl-3-oxo-2-phenyl- (CFUs) were determined by inoculating petri-plates actually contains
2,3-dihydro-1H-pyrazol-4-yl)amino)methyl)phosphonates(4h). Yield solidified agar medium of 100 μl from each dilution, and the rate of re-
(73%), IR (KBr) cm−1: 3745 (OH), 3493 (NH), 1680 (C_O), 1590 duction (R) of the treated samples was calculated as per the equation
(C_C, Ar.), 1276 (P_O), 773 (P\\C). 1HNMR (DMSO‑d6, 400 MHz): in relation to control (untreated).
1
HNMR (DMSO‑d6, 500 MHz): 2.398 (s, 3H, CH3), 3.11 (s, 3H, NH),
3.398 (s, 1H, CH), 6.767–6.783 (d, 3H, CHAr), 6.997–7.017 (dd, j = B−A
R ð%Þ ¼  100
34.5, 6H, CHAr), 7.281–7.364 (m, 7H, CHAr), 7.491–7.522 (m, 2H, CHAr), B
9.366 (s, 3H). 13CNMR (DMSO‑d6 500 MHz): 9.807, 112.983, 115.539,
117.094, 120.862, 124.248, 126.661, 129.132, 129.341, 134.750, wherever A, B are the CFU/mL for treated and untreated sample, respec-
145.633, 148.227, 151.623, 155.238, 159,969. LCMS, m/z tively after 16 h incubation [37].
(C30H28N3O6P) calcd, 557.54 [M]+; found, 557.1.
2.3.4. Preparation of carrageenan cryogel system and drug loading
2.3.1.9. Diphenyl((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol- Carrageenan solution (CAR; 2.5% wt./vol.) was prepared by dissolv-
4-yl)amino)(1H-indol-3-yl)methyl)phosphonate (4i). Yield (69%), IR ing 2.5 g of CAR in 100 mL distilled water at 90 °C for 60 min. The system
(KBr) cm−1: −3195 (-NH), 1682 (C_O), 116 (C_C, Ar.), 1299 was cross-linked by Glyoxal and three different concentrations were
(P_O), 748 (P\\C). 1HNMR (DMSO‑d6 500 MHz): −2.472 (s, 3H, used (0.1, 0.2, and 0.3 mL). The mixture kept under magnetic stirring
CH3), 3.095 (s, 3H, CH3), 3.322 (s, 1H, CH), 7.188–7.190 (m, 2H, CHAr), for 15 min at 70 °C. Since the pure CAR cryogel was found to be brittle
7.337–7.835 (m, 16H, CHAr), 8.401–8.430 (d, 2H, CHAr), 9.762 (s, 1H, and weak, cellulose nanofibrils (CNF, 2.5% w/v) was used to reinforce
NH), 11.48 (s, 1H, NH). 13CNMR (DMSO‑d6 500 MHz): 9.909, 111.745, the formed cryogel and provide mechanical strength. A definite volume
115.900, 118.563, 120.472, 121,978, 122.445, 123.879, 124.706, CNF was added to the CAR solution. The total volume of the sample was
126.262, 12.960, 131.316, 135.038, 137.2231, 150.833, 152.374, kept constant at 20 mL. The formulations of different samples are
160.360. LCMS, m/z (C32H29N4O4P) calcd, 564.58; found, 565.50 presented in Table 1. The resultant mixture was submitted to ultra-
[M + 1]+. sonication for 5 min to remove air bubbles. Then, the samples were fro-
zen in refrigerator overnight −80 °C and finally, the sample was dried at
2.3.1.10. Diphenyl((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H- −60 °C and 0.113 mbar using Labconco FreeZone 2.5 Freeze Dryer oper-
pyrazol-4-yl)amino)(3-hydroxynaphthalen-2-yl)methyl) ating. For further preparation and characterization, the resultant sam-
phosphonate (4j). Yield (68%), IR (KBr) cm−1: −3681 (NH), 1634 ples have been maintained in a desiccator.
(C_O), 1584 (C_C, Ar.), 1147 (P_O), 747 (P\\C). 1HNMR (DMSO‑d6 For the drug loading experiment, drugs with high biological efficacy
500 MHz): −2.428 (s, 3H, CH3), 3.224 (s, 3H, NH), 3.308 (s, 1H, CH), (4f, 4 g, and 4 h) was loaded to the optimized CAR/CNF cryogel sample
7.157–7.582 (m, 17H, CHAr), 7.854–7.933 (m, 2H, CHAr), 8.076–8.105 (CAR-7) and coded CAR-9, CAR-10, CAR-11, respectively. Drug solution
(d, 2H, CHAr), 10.648 (s, 1H, NH), 14.998 (s, 1H, OH). 13CNMR was prepared by dissolving 1 g of the solid drug in 50 mL of β-
(DMSO‑d6 500 MHz): −9.784, 109.901, 114.033, 119.208, 119.569, cyclodextrin solution (1% w/v in distilled water) and kept under mag-
123.363, 124.957, 127.241, 127.476, 127.753, 129.001, 129.123, netic stirring for 15 min then exposed to sonication for 10 min so that
131.953, 133.365, 134.158, 149.300, 154.138, 159.275, 160.941. LCMS, every 5 mL solution contains 100 mg of active drug. β-cyclodextrin is
m/z (C34H30N3O5P) calcd, 591.60; found, 590.0 [M-1]−.

2.3.2. Biological activity: qualitative screening of α-aminophosphonate Table 1

Formulation of carrageenan cryogel system.
derivatives
The prepared samples (5 mg) were dissolved in 2 mL of dimethyl Sample code Volume (mL)
sulfoxide (DMSO) and 100 μL (containing 250 μg) were used in the CAR CNF H2O Drug/cyclodextrin Glyoxal
test. The agar cup platform system was used to analyze the antimicro- (2.5% w/v) (2.5% w/v) (mL) (mL)
bial activity of various samples. The evaluation was carried out against CAR-1 15 – 5 0 –
the most pathogenic microbes such as S. aureus -, E. coli, C. albicans CAR-2 15 – 5 0 0.1
and A. niger. Nutrient agar plates with 0.1 mL of 105–106 cells/mL for CAR-3 15 – 5 0 0.2
bacteria and yeast have been extensively seeded uniformly. For the CAR-4 15 – 5 0 0.3
CAR-5 12.5 2.5 5 0 –
evaluation of the antifungal activities, a Czapek-Dox agar plate seeded
CAR-6 12.5 2.5 5 0 0.1
with 0.1 mL fungal inoculum was used. Though, in the media, the gel CAR-7 12.5 2.5 5 0 0.2
cutter (Cork Borer) was created in sterile condition by a hole (1 cm in CAR-8 12.5 2.5 5 0 0.3
diameter). Afterward, a drop of melted agar was dropped into the CAR-9 12.5 2.5 0 5 0.2
hole and made a simple layer solidified. Plate was therefore maintained CAR-10 12.5 2.5 0 5 0.2
CAR-11 12.5 2.5 0 5 0.2
for about 2–4 h at low temperature (4 °C) so that maximal diffusion
 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107 99

a cyclic oligosaccharides with a hydrophilic outer surface and a lipo- antimicrobial activity of the compounds was qualitatively and quantita-
philic central cavity [38]. The presence of β-cyclodextrin in the solution tively assessed and the most active compounds were selected for further
not only improved the solubility of the hydrophobic drug in water but application. In addition, the formation of cryogel system based on carra-
also increased its compatibility with the hydrophilic delivery system geenan was investigated as a delivery vehicle for the active compounds.
(CAR/CNF). The as synthesized drug was completely enclosed in β- The optimum conditions of cryogel formation using freeze-drying process
cyclodextrin hydrophobic cavity and will be referred in the manuscript were established and its physical and mechanical properties were investi-
as drug solution. The release profile and biological activity of such sam- gated. Three selected compounds were loaded to the cryogel and their re-
ples were investigated and reported in the results section. lease profile and antimicrobial activity were explored. The results and
discussion of the current study were presented in the above-mentioned
2.3.5. Physical, mechanical and biological characterization of CAR cryogel order. So as to integrate of mono α-aminophosphonates 4, the three pre-
Brunauer–Emmett–Teller (BET) analysis using Nova LX was used to cursors, aldehydes 1, 4-aminoantipyrene 2, and triphenylphosphite 3 in
assess surface area of the formed cryogels. In deionized water, a swell- acetonitrile were reacted with the aid of Lewis acid catalyst (10 mol%) of
ing measurement for the acquired cryogels was calculated by immers- LiClO4 (Scheme 1). The reaction continued for 5 days with high separated
ing the samples at room temperature for 24 h in order to establish the yields. The molecualr structures of α-aminophosphonates (4a–j) were af-
swelling equilibrium. Samples were then regularly collected, dried and firmed by IR, 1H NMR, 13C NMR and mass spectral analysis (for details cf.
weighed again. Before assessing, the excess of water has been removed the Experimental data).
from the surface of swelled cryogel sample via filter paper. Then, the The plausible mechanism for the formation of 4 is depicted in Scheme
swelling ratio according to Eq. [1] has been determined. 2. Firstly, the reaction is initiated when the catalyst (LiClO4) activates the
carbonyl group, which consequently condensates the starting aldehydes1
wet weight−dry weight with amine 2 to give Schiff base C. Afterwards, the nitrogen atom of Schiff
Swelling Ratio ¼  100% ð1Þ
dry weight base offers its pair of electrons to create a coordinate bond with LiClO4.
This coordination makes nitrogen positively charged and acquires a par-
The amount of water uptaked by the cryogel has been calculated in tial positive charge on sp2 carbon. Finally, the phosphorus lone pair of
accordance with Eq. (2). electrons phosphite 3 couples with the partially positive carbon atom
followed by addition of water and protonation of nitrogen and elimina-
wet weight tion of phenol to give α-aminophosphonates 4.
water uptake ¼  100% ð2Þ
dry weight
3.2. Biological activity of synthesized of α-aminophosphonate compounds
Each analysis was repeated three times in order to provide us as an
efficient way to improve test accuracy. 3.2.1. Qualitative screening via disc diffusion method
The synthesized cryogels were formed into a flat cylindrical plastic Among all organophosphorus derivatives, α-Aminophosphonates
container for compressive processing. Cylindrical cryogel's top and bot- are widely investigated due to its promising biological activity. They
tom sides are cut with a clean and sharp blade. A compressive load of have been used as antiviral, antimicrobial, antifungal, anticancer and
0.5 N using a rate of 5 mm/min in an Instron Series IX was utilized for in different applications. In the current work, the antimicrobial activity
obtaining the curves of compressive stress. The modulus from Young of the as prepared α-Aminophosphonates were tested via the agar
is seen by measuring the compressive stress train curve slope at a strain plate method against Gram-negative bacteria, E. coli; Gram-positive
of 1%. A Field Emission Scanning Electron Microscope (FESEM; bacteria, S. aureus; a fungus, A. niger; and a yeast, C. albicans. The zone
FEIVerios460L) was utilized to examine the morphology of the formed of inhibition is observed in Fig. 1 and reported in Table 2. The tested
cryogels. Duplicate was carried out to determine the in-vitro release compounds showed activity against C. albicans and A. Niger. Compounds
studies of the investigated compounds (4f, 4g, and 4h) loaded CAR 4f, 4g, and 4h presented the highest antimicrobial efficiency with inhibi-
cryogels. Shortly, in a beaker containing 100 ml of phosphate buffer sa- tion zone ranges from 13 to 19 mm on the agar plates.
line (pH 5.5), 100 mg of freeze-dried samples were placed.
The glass beaker was placed in a mechanical shaking water bath 3.2.2. Determination of antimicrobial efficacy of selected drugs via colony
(50 cycles/min) at 37 °C. At selected time intervals (between 30 min forming unit (CFU) method
and 480 min) samples were withdrawn and replaced with fresh phos- It is well known that bacterial infections from S. aureus and E. coli are
phate buffer. In a mechanical water-shaking bath (50 cycle/min) at most likely to happen in hospitals and severely affect patients with
37 °C, the samples were placed the glass beaker. Then, 2 mL of the solu- wound healing problems [40]. Since one of the targets of this research is
tion loaded drug was taken and replaced by fresh phosphate buffer at to develop a delivery system for wound dressing applications, further in-
specified time intervals (between 30 min and 480 min). Ultimately, vestigation of the antimicrobial efficiency of some selected compounds is
UV–vis spectroscopy (Shimadzu UV-1601PC Spectrophotometer, En- performed. Quantitative investigation of the antimicrobial activity of com-
gland) used for calculating the amount of drug released. pounds 4f, 4g, and 4h was executed against S. aureus and E. coli using the
viable cell counting method. Number of survival colonies of different bac-
3. Results and discussion terial dilution was reported in Tables 3 and 4 and the resultant agar plates
are presented in Figs. 2 and 3 for S. aureus and E. coli, respectively.
3.1. Chemistry Two different concentrations (15 and 30 mg) form the as prepared
compounds were used for each 10 mL of bacterial solutions. The microbial
Numerous synthesis courses were accounted for α- inhibition was dictated by figuring the decrease development rate for of-
aminophosphonates, but the one pot Mannich-type procedure of carbonyl fered tests in connection control (untreated) sample. It is clear from the
mixes, amines, and trialkyl/triaryl phosphites in the presence of a Lewis results that lower number of bacterial colonies as detected at upon the
acids remains the most proficient, straightforward, general, and high yield- use of high concentration of active compounds (30 mg/10 mL). Addition-
ing strategy [39]. The current research is using such methodology to pre- ally, the tested drugs exhibited higher efficacy against S. aureus than
pare new α-aminophosphonates (total ten compounds) with expected E. coli. In the case of S. aureus, all compounds were found to be bactericidal
biological activity. 4-Aminoantipyrine was used as a base amine and differ- at 105 dilution. Only 4f and 4h at high concentration (30 mg/10 mL) are
ent aldehydes were used in the synthesis procedure. The chemical struc- bactericidal at higher bacterial concentration (102 dilution). On the
ture of the prepared compounds was investigated and interpreted by other hand, only 4f (30 mg/10 mL) was found to be bactericidal at higher
several techniques including NMR, FTIR, and Mass spectrometry. The bacterial concentration (102 dilution).
100 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107

Scheme 1. Synthesis of α-aminophosphonates from 4-aminoantipyrene, different aldehydes and triphenylphosphite

3.3. Preparation of carrageenan cryogels structure than the aerogels but higher than that of xerogels. The exis-
tence of such pores within the structure of the cryogels facilitate its
Sponge structures such as; aerogels, xerogels and cryogels have been swelling and offer a reliable hosting space for loading of active materials
widely used as drug delivery vehicles [41]. These structures are noted as such as metal nanoparticles and antibiotics [26]. Herein, cryogel was
highly to low porous according to the fabrication method. Cryogels are formed through three steps; sol followed by gel and ultimately cryogel
obtained with the aid of freeze-drying technique retain lower porous formation. The sol process involved the mixing two polymers;

Scheme 2. Plausible Mechanism for α-Aminophosphonates 4

 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107 101

Fig. 1. Antimicrobial screening of the as prepared compounds via zone of inhibition assessment against S. aureus, E. coli, C. albicans and A. niger.

carrageenan (CAR) and cellulose nanofibrils (CNF) under mechanical became more resistant to squishing by adding glyoxal to the samples as
stirring to form gel. The formed mixture was transformed to gel after a cross-linker. Samples; CAR-2, CAR-3, and CAR-4 contain 0.1, 0.2, and
definite time and microporous texture is developed with the aid of dif- 0.3 mL of glyoxal, respectively. It is clear from the photos that CAR-4 ex-
ferent amounts (0.1, 0.2, and 0.3 mL) of crosslinking agent (glyoxal). perienced more shrinkage than CAR-2 and CAR-3. This may be attributed
The second step is to freeze the produced mixture at −60C to keep to the strong intermolecular cross-linking that occurred inside the carra-
the ice crystals zone of the mixture without degradation. The third geenan chains upon applying higher concentration of glyoxal. Upon addi-
step is drying at very low temperature and under pressure to evaporate tion of CNF to the cryogel system, the apparent brittleness improved to
the entrapped solvent from the formed pores but with no degradation large extent and samples CAR-6, CAR-7, and CAR-8 showed higher resis-
or agglomeration. Overall, freeze-drying aimed to prepare the cryogel tance to squishing when pressed. CAR-8 showed similar behavior as its
without destroying the microstructure of the pore walls [42]. At the counterpart (CAR-4) due to the high concentration of glyoxal. This im-
end of drying, cryogel with definite shapes can be obtained. provement in the apparent strength of the cryogel could be attributed
Fig. 4 shows photo images for the as prepared carrageenan cryogel. By to the formation of strong covalent bonds between CAR and CNF on
visual inspection, pure carrageenan (CAR-1) tends to form a very brittle micro chain level which consequently lead to stronger interconnected
cryogels. However, the apparent brittleness improved and cryogel network between CAR and CNF [38].

Table 2
Inhibition zone of as prepared aminophosphonate compounds.

Serial no. Compound Clear zone (mm)

S. aureus E. coli C. albicans A. niger

4a 0 13 15 0
1
4b 14 16 13 14
2
4c 13 0 14 18
3
4d 15 0 12 16
4
4i 15 0 15 13
5
4e 0 9 16 14
6
4f 13 15 14 0
7
4g 16 13 13 12
8
4h 19 15 16 15
9
4j 12 12 14 16
10
102 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107

Table 3
Number of survival colonies of different dilution of S. aureus upon treatment with 4f, 4g, and 4f.

Compound Concentration mg/10 mL Number of colonies at each dilution

101 102 103 104 105 106 107

4f 15 29 5 3 0 0 0 0
30 0 0 0 0 0 0 0
4g 15 32 14 11 22 5 0 0
30 22 7 2 0 0 0 0
4h 15 87 66 36 2 0 0 0
30 12 0 0 0 0 0 0
Control nc nc nc nc 487 105 27

Nc = couldn't count.

3.4. Physical, mechanical, biological characterization of carrageenan the swelling percentage increased up to 70% of its original weight. Nev-
cryogels ertheless, utilizing higher amount of glyoxal (0.3 mL) leads to a sharp
decrease in swelling (around 35%). This could be attributed to the
3.4.1. Surface area and swelling properties high crosslinking degree achieved by such glyoxal concentration,
After observing the apparent brittleness of the as prepared cryogel, it which consequently lead to shrinkage and blockage of microporous
is necessary to determine the surface area and swelling properties to de- structures. It is worthy to mention that these microporous are consid-
fine the potential of these cryogels for drug loading. As shown in Fig. 5, ered as hosting cavity for holding the water and liquids [45]. Via addi-
cryogel formed from pure CAR has very low porosity (less than 35%) tion of CNF to CAR solution, it is observed that the value of swelling
which depicts that CAR is not able to form interconnected network. property reaches around 35% (sample CAR-5), which is considered as
For this interconnected network formation, crosslinking agent should high value comparing to that of pure CAR. However, the cryogel par-
be added to CAR solution prior to cryogel formation [26,43]. Therefore, tially disintegrated and couldn't keep its structural integrity upon swell-
sample CAR-2 that means the cross-linked CAR cryogel with glyoxal ing, however, it performed better that CAR-1 due to the intermolecular
(0.1 mL) has significant high porous value when compared with the hydrogen bonding between CAR and CNF. Upon incorporation of differ-
value of uncross-linked cryogel (CAR-1). With increasing the ent concentrations of glyoxal; (0.1 mL and 0.2 mL) of glyoxal to CAR and
crosslinking agent to 0.2 mL, the microporous structures value sharply CNF mixture the value of swelling sharply increased to exceed 75% for
increases to more than 50% (CAR-3) which depicts that glyoxal as CAR-7. Meanwhile, the value of swelling is marginally decreased to
crosslinking agent has the ability to form strong bonds between CAR about 65% when higher glyoxal 0.3 mL was added (CAR-8). Based on
chains forming interconnected network. However, increasing the con- the aforementioned data, the results of swelling percentage is in accor-
centration of glyoxal to 0.3 mL as shown in CAR-4, the microporous dance with that of BET examination. The presence of glyoxal as a cross
structures reduces to be lower than CAR-3 which may be attributed to linker is more favorable at 0.2 mL of to gain a good cryogel of CAR and
the blocking effect of crosslinking to most of the interconnected net- the augmentation of CNF into the system improved its water holding ca-
work that formed between CAR chains. pacity [46].
On the other hand, addition of CNF to CAR enhances the formation of
these microporous structures for CAR as shown in CAR-5 (about 45%) 3.4.2. Mechanical properties
comparing to CAR-1 (in absence of crosslinking agent). Samples CAR-6 The mechanical compressive strength has been examined to affirm
and CAR-7 contain CNF and of 0.1 mL and 0.2 mL of glyoxal solution, re- the effect of CNF and glyoxal as reinforcement agent and crosslinking
spectively, showed improved porosity up to 80%. Obviously, the agent, respectively, on the mechanical strength of CAR cryogel.
crosslinking agent is essential to enhance the formation of microporous Fig. 6 displays the compressive strength in terms of strain and stress
structures and maximize its value [44]. However, using crosslinking factors of native, cross-linked, and CNF reinforced CAR cryogel. It is
agent above 0.2 mL as investigated in CAR-8, results in sharp decrease observed that uncross-linked CAR/CNF (CAR-5) cryogel shows better
porosity percent (68%) for the same reason mentioned before for CAR-4. mechanical properties compared with the uncross-linked native CAR
The swelling performance of the as prepared cryogels was investi- (CAR-1). However, the mechanical properties of both CAR and CAR/
gated by monitoring the weight percentage increase upon immersion CNF are very low due to their brittleness. On the other hand, the
in deionized water for 24 h at room temperature. It is observed that cross-linked samples (CAR-2, CAR-3, CAR-4) showed enhanced me-
pure CAR cryogel (CAR-1) is partially disintegrated and the structure chanical strength compared with the native CAR cryogel (CAR-1). This
could not hold its integrity. By adding glyoxal as a crosslinking agent could be attributed to the presence of crosslinking agent that creates
(0.1 mL, 0.2 mL) as shown for the samples coded with CAR-2 and strong covalent bonds between CAR micro chains. In addition, the
CAR-3, respectively, the cryogels were able to keep its integrity and cross-linked CAR/CNF (CAR-6, CAR-7, CAR-8) exhibited higher

Table 4
Number of survival colonies of different dilution of E. coli upon treatment with 4f, 4g, and 4f.

Compound Concentration mg/10 mL Number of colonies at each dilution

101 102 103 104 105 106 107

4f 15 3 2 0 0 0 0 0
30 2 0 0 0 0 0 0
4g 15 Nc nc nc nd 91 42 17
30 nc nc nc 598 76 23 0
4h 15 Nc 384 175 18 7 6 0
30 nc 601 72 11 2 0 0
Control nc nc nc nc 371 103 48

Nc = couldn't count.
 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107 103

Fig. 2. Agar plates of S. aureus survival colonies upon treatment with different concentrations (15 and 30 mg/10 mL bacterial solution) of 4f, 4g, and 4f compounds.

Fig. 3. Agar plates of E. coli survival colonies upon treatment with different concentrations (15 and 30 mg/10 mL bacterial solution) of 4f, 4g, and 4f compounds.
104 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107

Fig. 4. Cryogel system made with different formulation. Pure carrageen (CAR-1); carrageenan/CNF (CAR-5); carrageenan with glyoxal (CAR-2, 0.1 mL; CAR-3, 0.2 mL; and CAR-4, 0.3 mL);
and carrageenan/CNF with glyoxal (CAR-6, 0.1 mL; CAR-7, 0.2 mL; and CAR-8, 0.3 mL).

mechanical properties than cryogel formed in absence of CNF. It could with using moderate concentration of the crosslinking agent; 0.2 mL
be assigned to the formation of stronger interconnected network be- as in sample (CAR-7).
tween CAR and CNF [38]. Therefore, CNF acquired strong reinforcement
and enhancement the mechanical properties of CAR cryogel. This made 3.4.3. Topographical structure
the cryogel samples more resistive to compressive loads during testing. Fig. 8 shows SEM micrographs of the morphological surface of se-
This improvement in mechanical properties makes the CAR cryogel lected samples, namely, carrageenan (CAR-1) and carrageenan/CNF,
have potential candidate for drug delivery application, particularly 0.2 mL glyoxal (CAR-7). It is clear from Fig. 8-A that the surface of

Fig. 5. BET and swelling, properties of the formed cryogel. Pure carrageen (CAR-1);
carrageenan/CNF (CAR-5); carrageenan with glyoxal (CAR-2, 0.1 mL; CAR-3, 0.2 mL; and Fig. 6. compression test for CAR cryogel. Pure carrageen (CAR-1); carrageenan/CNF (CAR-
CAR-4, 0.3 mL); and carrageenan/CNF with glyoxal (CAR-6, 0.1 mL; CAR-7, 0.2 mL; and 5); carrageenan with glyoxal (CAR-2, 0.1 mL; CAR-3, 0.2 mL; and CAR-4, 0.3 mL); and
CAR-8, 0.3 mL. carrageenan/CNF with glyoxal (CAR-6, 0.1 mL; CAR-7, 0.2 mL; and CAR-8, 0.3 mL).
 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107 105

pure carrageenan has resulted in lamellar structure with no pores. How- tension might be associated with the stabilization of cyclodextrin com-
ever, incorporation of CNF and crosslinking with glyoxal lead to the for- plex arrangement. This complexation doesn't include formation of cova-
mation of porous structures as displayed in Fig. 7-B (higher lent bonds. This means that the enclosed molecules situated inside the
magnification is presented in B-1 and B-2). This can be attributed to cavities are in a dynamic balance with the free medication ones in solu-
the fact that carrageenan lean towards formation of gels quickly after tion [52]. Fig. 8(i–iii) shows the solubility of the compound 4g in;
it dissolves [47]. Moreover, gels with interconnected pores and pore i) DMSO; ii) β-cyclodextrin solution; and iii) distilled water. It is notice-
walls being partly sheet-like are most likely to form because of the able that the compound precipitate in the vial and not soluble at all in
freeze-drying process [48]. However, the presence of CNF and glyoxal distilled water. The solubility greatly improved in cyclodextrin solution
in the blend tends to form interconnected network with the carra- due to the inclusion in its hydrophobic cavity.
geenan molecular chains, which may delay the gel formation, and con- β-Cyclodextrin not only improved the solubility of the as prepared
sequently form pores. The presence of such porous structure makes the aminophosphonate drugs in water but also enhanced its compatibility
investigated sample a candidate for drug loading applications. with the hydrophilic cryogel system; CAR/CNF. Fig. 8(A-C and a-c) rep-
resents CAR-9, loaded with 4f; CAR-10, loaded with 4g; and CAR-11,
3.4.4. Drug loading and in-vitro evaluation of drug release loaded with 4f while a, b, c are their swollen equivalents. It is clear
Upon the conclusion of the BET, swelling capacity, and mechanical that the as prepared cryogels were able to keep their structural integrity
properties results, CAR-7 was selected for drug delivery application. and remained intact upon swelling during the release measurement.
The release was performed in phosphate buffer saline (pH 5.5). Thanks This behavior could be attributed to the efficient intermolecular cross-
to large BET value (75%), swelling percentage (80%) and relatively high linking of carrageenan chains and intramolecular cross-linking of carra-
mechanical properties of CAR-7. The uncross-linked CAR/CNF cryogels geenan and CNF molecular chains.
were excluded from the in-vitro release studies because of the lack of For drug loading experiment, exactly 5 mL of each drug solution
structure integrity upon swelling. This is attributed to the hydrophilicity (contains 100 mg solid drug) was loaded to the CAR/CNF polymer solu-
of the two base polymers (CAR and CNF) thus would rapidly collapse in tion blend before the formation of the cryogel (Fig. 8). The concentra-
aqueous medium leading to a fast release of drug molecules [49]. tion of the released drug is determined using UV spectrophotometer
Three model synthesized aminophosphonate drugs; 4f, 4g, and 4h at 315, 325 and 360 nm for 4f, 4g, and 4h respectively. Calibration was
were selected for further evaluation. As know, these drugs are hydro- obtained by using standard samples. Fig. 9 shows the release profile of
phobic and they were suspended in β-cyclodextrin solution before the these drugs (loaded to CAR-7) over time.
addition to CAR/CNF solution. These three nominated drugs that loaded As can be seen in Fig. 9, the release of three drugs from microstruc-
CAR/CNF cryogel are coded CAR-9, CAR-10 and CAR-11, respectively in tural CAR matrices is initially fast followed by a sustained controlled
Table 2. β-cyclodextrin is perceived as a valuable device for improving after 30 min. The change in the profile of the three drugs could be re-
the stability and dissolvability issues of hydrophobic materials [50]. It lated to the improved affinity between cryogel components and drug
is cyclic oligosaccharides that comprise of 6–8 dextrose units framing due to the presence of β-cyclodextrin. There is a marginal difference
a donut structure. β-cyclodextrin naturally acquired a lipophilic internal in the release profile of the tested drugs due to the similarity in the
cavity and a hydrophilic external surface [51]. In watery arrangements, structure as well as hydrophobicity nature. However, the sustained re-
β-cyclodextrin structure complexes with hydrophobic moieties where lease was observed over longer time for CAR-11. This behavior may be
such molecules displace the water particles situated in the internal cav- explained by the proper intramolecular crosslinking degree within
ity. Few powers including van der waals and hydrophobic interactions, CAR chains as well as intermolecular crosslinking between CAR, CNF,
hydrogen bonding, ease structural strains, and changes in surface and β-cyclodextrin. Additionally, hydrogen bonding may occur

Fig. 7. SEM micrographs of Carrageenan cryogels, A) carrageenan (CAR-1); B) carrageenan/CNF, 0.2 mL glyoxal (CAR-7); B-1 and B-2 are higher magnification of (CAR-7)
106 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107

Fig. 8. Solubility of aminophosphonate drug (4f) in i) DMSO; ii) cyclodextrin solution; and iii) distilled water. A) CAR-9, loaded with 4f; B) CAR-10, loaded with 4g; C) CAR-11, loaded with
4f; while a, b, c are their corresponding swollen samples.

The antimicrobial activities were performed using the colony

forming unit. Results in Table 5 show the CFU of different samples and
the control (S. aureus) at different dilutions (from 10−1 to 10−7). It
has been found that sample CAR-9 loaded with 4f showed the highest
reduction (with reduction percent of 99.94%) followed by sample
CAR-11 loaded with 4h (with reduction percent of 99.93%) and finally
CAR-10 loaded with 4g (with reduction percent of 99.29%) as compared
to the control sample (S. aureus). The 4th dilution was used for
comparison.

4. Conclusion

One pot and three-component reaction of 4-aminoantipyerene with

different aldehydes was implemented to prepare α-aminophosphonate
compounds. Spectral (IR, MS, and 1H NMR) studies proved successful
preparation of total ten derivatives. Compounds (4f-h) were found bio-
logically active against S. aureus, . coli, C. albicans and A. niger and were
selected for further drug delivery studies. The incorporation of CNF
and glyoxal was very essential for achieving proper mechanical proper-
Fig. 9. Release profile of 3 4f; 4g; and 4h compounds from CAR-9; CAR-10; and CAR-11 ties for drug delivery applications since the pure CAR cryogel is very
cryogel over time. fragile. The optimum conditions of formation of CAR based cryogel sys-
tem (16% CNF and 0.2 mL/15 mL polymer blend) were presented by
between -OH, -COOH of CNF and CAR with the functional groups of the sample CAR-7, which demonstrated highest mechanical compressive
4 h drug since it has two hydroxyl groups in base aldehyde structure. strength, porosity (80%), and high swelling capacity (75%). Sustainable
release performance was observed for the CAR-9, CAR-10, and CAR-11.
Negligible differences in antimicrobial activities of the aforementioned
3.4.5. Determination of antimicrobial efficacy of drug loaded cryogel via drug loaded cryogels against S. aureus were observed. The results
colony forming unit (CFU) method against Staphylococcus aureus showed that CAR-9 has highest reduction in number of bacterial colo-
The sample coded with CAR-7 has been selected to be loaded with nies (99.94%) followed by CAR-11 (99.3%), and finally CAR-10
different types of the synthesized drugs; 4f, 4g and 4h, these formed (99.29%) when compared to the control. In conclusion, the as prepared
cryogels are coded CAR-9, CAR-10 and CAR-11 respectively. Then, α-aminophosphonates showed high potential of new antimicrobial
their antimicrobial activity against S. aurous was tested and results are agents and the investigated carrageenan/cellulose nanofibrils cryogel
illustrated in Table 5. system could be used in wound care field.

Table 5
Antimicrobial Activity of CAR-9, loaded with 4f; CAR-10, loaded with 4g; and CAR-11, loaded with 4f against S. aureus using CFU method.

Dilutions Colony forming units (CFU/mL) Control

CAR-9 (loaded with 4f) CAR-10 (loaded with 4g) CAR-11 (loaded with 4h)

10−1 411 ∞ ∞ ∞
10−2 372 ∞ 541 ∞
10−3 201 ∞ 332 ∞
10−4 27 321 31 ∞
10−5 8 53 10 ∞
10−6 3 10 0 317
10−7 0 1 0 45
 D.A. Elsherbiny et al. / International Journal of Biological Macromolecules 163 (2020) 96–107 107

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