Preparation, Characterization of New Antimicrobial Antitumor Hybrid Semi-Organic Single Crystals of Proline Amino Acid Doped by Silver Nanoparticles

Proline is water soluble amino acid extensively used in drug delivery systems. Compounds of cobalt (Co) transition metal have potent antimicrobial and anticancer activities. However, a drug delivery system combining proline cobalt is not reported yet. For the first time, new hybrid semi-organic single crystals of proline cobalt chloride (PCC) are prepared. The novelty of the article is also that single crystal proline cobalt chloride showed potent antimicrobial and antitumor activity. Doping of PCC by Ag0NPs significantly increased these biological activities. The anisotropic magnetic properties of single crystals can mitigate the cytotoxicity of Ag0NPs on normal cells. Silver nanoparticles (Ag0NPs) improved the crystal habits and physicochemical properties. Ag0NPs showed the best performance, paramagnetic materials n-type semiconductors due to delocalized excess electrons of Ag0NPs incorporated in the crystal lattice interstitially. Crystals have high absorptivity for UV-radiation electromagnetic radiation. Ag0NPs enhanced AC electrical conductivity up to 2.3 × 104 Ω cm−1 due to high electron density. Proline doped crystals are obtained in good purity as triclinic unit cell with having anisotropic magnetism. PCCAg0NPs crystal exhibited: high antimicrobial activities to various bacterial and fungal species, inhibition zone (mm): 21, 25, 24, 26, 30, 28, 12, and 46 for S. aureus, E. faecalis, S. typhi, E. coli, P. aerugino, K. pneumoniae, A. braselienses, and C. albicans, respectively, in comparison to ciprofloxacin antibiotic (23, 0, 26, 26, 25, 0, 0, 0) for the same tested species, respectively; higher cytotoxicity against breast cancer cells (IC50 22.1 μM) than the reference drug cisplatin (IC50 11.7 μM); and lower cytotoxicity to normal healthy lung cells MRC-5, (IC50 145.5 μM) than cisplatin (IC50 30.2 μM). Hence, this crystal is a candidate for chemotherapy of breast cancer.


Introduction
Single crystals in literature are described as polar crystals, thermoelectric materials that generate electricity on heating that change intrinsic magnetic moments and permittivity; tiny small sensors in electronic and power generators convert chaotic waste heat into useful electric work [1,2]. They store electrical energy after removal of an applied electric field as charge-reservoirs parallel plate capacitors; piezoelectric materials applied in automobiles electronics, touch screens of laptop and mobile phones; microwave filters, and energy storage systems [3,4]. In electric fields, the electron clouds in atoms polarize. The dielectric constant or refractive index is affected by the frequency of absorbed quantized energy UV-Vis.; and absorb UV-Vis. of sunlight generating electron-hole pairs [5]. The current flow depends on photon energy. Polarization in electric fields is controlled by dielectric constant; and AC conductivity measures macroscopic dielectric properties and polarization [5]. Appropriate salts weights are mixed in stoichiometric molar ratio: (glycine or proline) 1−x (CoCl 2 ) x (x is dopant weight percent of either CoCl 2 in absence and presence of Ag 0 NPs). Glycine single crystal is grown as a control single crystal.
Single crystals are grown following a slow evaporation method [12], at optimum experimental conditions: temperature 25 • C ± 0.1, pH 5.5 and 100 rpm agitation speed for 2 h. The salts mixture is continuously agitated in deionized water green nontoxic solvent until attaining a homogeneous saturated solution at the same temperature. The saturated solution is filtered and left covered with porous filter paper. Crystal nucleation and growth are allowed through slow water evaporation. Pink colored single crystals: proline cobalt chloride (PCC) in the presence of 0.15 wt.% Ag 0 NPs are harvested after three weeks of representative visual appearances as shown in Figure 1 in comparison to glycine cobalt chloride (GCC). solution is filtered and left covered with porous filter paper. Crystal nucleation and growth are allowed through slow water evaporation. Pink colored single crystals: proline cobalt chloride (PCC) in the presence of 0.15 wt.% Ag 0 NPs are harvested after three weeks of representative visual appearances as shown in Figure 1 in comparison to glycine cobalt chloride (GCC). High quality crystals have a large size, and perfect octahedral (Oh) geometry with defined edges. Crystals containing Ag 0 NPs have a more intense pink color, suggesting applications as new colored materials in the second harmonic generation.

Characterization of Single Crystals
Crystals are characterized using different spectroscopic methods of analysis. Carbon, hydrogen, and nitrogen CHN elemental analysis (EA) is determined using Malvern analytical elemental analyzers. The content of Co(II) ion is determined via several digestion decompositions in aqua-regia to dissolve organic matter. Cobalt residue is dissolved in double distilled water and is determined using a Shimadzu 6650 atomic absorption spectrophotometer.
Powder X-ray diffraction at 2θ range 5-80° with Cu-Kα X-ray (λ 1.54 Å) radiation source. Density, ρ, is determined by floatation technique in a saturated solution of NaCl, KBr and benzene separately. The number of formula units per unit cell (Z) is calculated by using the equation [17,18]: where V is volume of unit cell, and N is Avogadro's number. X-band electron spin resonance spectra at room temperature using a reflection (JES-RE1X ESR. ESR spectrometer) at 9.43 GHz in cylindrical resonance cavity, 100 kHz modulation, 5 mW electric power and LMR Gauss meter control applied magnetic field.
AC electrical conductivity of single crystal sample is measured using four probes Agilent 4294 A Impedance Bridge applying sine AC signal, 10 amplitude. Thin gold layers (10 nm) are deposited on two opposite sides of the pellet sample by thermal evaporation High quality crystals have a large size, and perfect octahedral (Oh) geometry with defined edges. Crystals containing Ag 0 NPs have a more intense pink color, suggesting applications as new colored materials in the second harmonic generation.

Characterization of Single Crystals
Crystals are characterized using different spectroscopic methods of analysis. Carbon, hydrogen, and nitrogen CHN elemental analysis (EA) is determined using Malvern analytical elemental analyzers. The content of Co(II) ion is determined via several digestion decompositions in aqua-regia to dissolve organic matter. Cobalt residue is dissolved in double distilled water and is determined using a Shimadzu 6650 atomic absorption spectrophotometer.
Powder X-ray diffraction at 2θ range 5-80 • with Cu-Kα X-ray (λ 1.54 Å) radiation source. Density, ρ, is determined by floatation technique in a saturated solution of NaCl, KBr and benzene separately. The number of formula units per unit cell (Z) is calculated by using the equation [17,18]: where V is volume of unit cell, and N is Avogadro's number. X-band electron spin resonance spectra at room temperature using a reflection (JES-RE1X ESR. ESR spectrometer) at 9.43 GHz in cylindrical resonance cavity, 100 kHz modulation, 5 mW electric power and LMR Gauss meter control applied magnetic field.
AC electrical conductivity of single crystal sample is measured using four probes Agilent 4294 A Impedance Bridge applying sine AC signal, 10 amplitude. Thin gold layers (10 nm) are deposited on two opposite sides of the pellet sample by thermal evaporation under vacuum 10 −5 mbar using Joule evaporator. Silver wire is glued on each deposit with silver lacquer.
Antimicrobial activity is determined using paper disk diffusion method against Grampositive bacteria: Staphylococcus aureus, Enterococcus faecalis; Gram-negative bacteria: Escherichia coli; Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae); and Fungi: Aspergillus brasiliensis; Candida albicans. Cytotoxic activity this metal complexes on cancer cell lines are screened using MTT assay against MCF-7 (human breast adenocarcinoma), and healthy MRC-5 human lung fibroblasts (control cell lines). Results of in vitro cytotoxic activity are compared with reference standard Cis-platin in terms of IC 50 .

Results and Discussion
The chemical composition and atomic percent are collected in Table 2. High atomic percent C, H, N, and O atoms indicated that proline and glycine amino acids are the mother materials for crystals [19]. Both proline and glycine have a white color. Doping of the crystal lattice of both proline and glycine by CoCl 2 produced optically active have high molecular weight (Mw.) single crystals of pink color. Ag 0 NPs intensified the pink color of PCC and increased Mw. up to 1051.54 g mol −1 forming a self-supramolecular assembled single crystal [20]. CoCl 2 incorporated into glycine and proline, forming a crystal with a 1:2 molar ratio. The crystals are stable, non-hygroscopic and soluble in water polar green solvent.
The infrared spectra of the crystals are compared with that of amino acids to deduce the intercalation mode. The charge transfer from ligand (proline or glycine) to Co(II) ion decreased the force constant of bond causing red shift of bond position and enhanced optical activity of crystals. Some blue shift occurs on back donation of the electron from Co(II) ion to the electron donor atom to reinforce the coordinate bond [21]. Assignments of main spectral vibrational band are given in Supplementary Information, Table S1.
FTIR spectral bands of proline are compared with that of single crystal to declare bonding mode with Co(II) ion. IR spectral bands of two PCC 2 crystals showed a ν COOH band at the 1730-1732 cm −1 range (Carbonyl group). The absence υ asy COO-is due to protonation on coordination to the Co(II) ion. υ NH and δ NH are red shift by 101-107 and 17-19 cm −1 , respectively, relative to proline. υ C-N and γ NH changed in shapes and positions, indicating the participation of a N atom in chelation. PCC showed a new band at 437-441 cm −1 (υ Co-N ). IR spectra explored amino acid is bidentate ligand coordinate Co(II) ion through N, O atoms, see Figure 2. nation on coordination to the Co(II) ion. υNH and δNH are red shift by 101-107 an cm −1 , respectively, relative to proline. υC-N and γNH changed in shapes and position cating the participation of a N atom in chelation. PCC showed a new band at 437-4 (υCo-N). IR spectra explored amino acid is bidentate ligand coordinate Co(II) ion t N, O atoms, see Figure 2.  Optical activity confirmed electronic spectral and magnetic properties of crystals investigated by using Nujol mull absorption spectroscopy at room temperature [23,24]. Calculated optical parameters: ligand field splitting and stabilization energy (CFSE), 10 Dq are collected in Table 3. Ligand field parameters for Co(II)-Racah inter-electronic repulsion parameter B : 595-753 cm −1 . The lowering B of free Co(II)ion complexation suggests orbital overlap and electrons delocalization on Co(II)ion. In nephelauxetic ratio, the β less than one indicating partial covalent bond "σ" between Co and amino acid [25]. The parameters of tetragonal distortion in crystals (Ds and Dt) and the crystal field parameter (Dq) are derived from the energy of different electronic transitions. Table 3. Electronic absorption spectral data λ max (nm) and effective magnetic values (µ eff 298 K) of crystals. Glycine-Co(II) The values of magnetic moment have B.M suggesting high spin distorted tetragonal geometry, (t 2g ) 5 (e g ) 2 configuration and 4 A 2g ground state. PCC displayed five absorption bands indicating axial distorted Oh symmetry around Co(II) ion [26] due to transitions were not observed in the spectra [27,28].
Thermal degradation confirmed the molecular structure and thermal stability. TGA and DTA thermograms of crystal are shown in  TGA showed weight loss of tested sample as a function of temperature or time. DTA measures temperature difference (∆T = T S − T R ) between the sample (S) and reference (R) materials at zero heat flow difference (∆H = H S − H R = 0).
TGA and DTA of proline and crystals showed distinguished coordination and stability ranges in peak temperatures and kinetic parameters. Shape index symmetry of peak "S" ratio of slopes of curve tangents at inflection points a/b depends on reaction order, n (1 • , 2 • order, etc.) and is determined from DTA, see Supplementary Information SI.1 [29].
Applying least square method, the plot (ln ∆T versus 1/T) is represented in Figure S1 and gave straight lines obeying Arrhenius relation. Activation energy E a of decomposition is calculated [30]. The TGA %wt.loss for decomposition steps is correlated to the proposed chemical formula, see Table 4.     TGA and DTA of proline and crystals showed distinguished coordination and stability ranges in peak temperatures and kinetic parameters. Shape index symmetry of peak "S" ratio of slopes of curve tangents at inflection points a/b depends on reaction order, n (1°, 2° order, etc.) and is determined from DTA, see Supplementary Information SI.1 [29].
Applying least square method, the plot (ln ΔT versus 1/T) is represented in Figure S1 and gave straight lines obeying Arrhenius relation. Activation energy Ea of decomposition is calculated [30]. The TGA %wt.loss for decomposition steps is correlated to the proposed chemical formula, see Table 4.
Proline showed 2.14% wt.loss at 199 °C, which caused a weak DTG peak at 66 °C corresponding to dehydration. Thermal degradation from 199-266 °C, 97.86% wt.loss due to complete decomposition is associated with broad DTG peak at 244.37 °C, in a narrow temperature range indicating rapid thermal decomposition. The corresponding Ea kJ/mol −1 and n values for two exothermic steps are 32.42, (1.05) and 12.88, (1.09), respectively [31]. Consecutive thermal degradation followed 1° order kinetic according to Scheme 1.

Figure 5.
Thermal gravimetric analysis and differential thermal analysis thermograms of PCCAg 0 NPs. PCC five degradation stages: 24.48% wt.loss is due to partial dehydration and removal six water molecules give strong DTG peaks, and Tmax 82 °C. Decomposition steps are at temperature ranges 192-302, 302-366 and 366-600 °C. In DTG weak, medium and strong peaks located at 214, 327 and 537 °C, respectively. Thermal decompositions steps suggested elimination: two coordinated H2O molecules, 2OH group and fraction residue 4 C atoms. The final degradation step showed wt.loss 13.02% at 600 °C, DTG peaks at 753 °C is due to release 1.0 mole H2(g) + 2.0 moles NH3 (Ea 122.22 kJ/mol, n 1.41), respectively. The final residue is CoO + 6C. Thermal decomposition pathway is represented in Scheme 2 [32].  PCC five degradation stages: 24.48% wt.loss is due to partial dehydration and removal six water molecules give strong DTG peaks, and T max 82 • C. Decomposition steps are at temperature ranges 192-302, 302-366 and 366-600 • C. In DTG weak, medium and strong peaks located at 214, 327 and 537 • C, respectively. Thermal decompositions steps suggested elimination: two coordinated H 2 O molecules, 2OH group and fraction residue 4 C atoms. The final degradation step showed wt.loss 13.02% at 600 • C, DTG peaks at 753 • C is due to release 1.0 mole H 2(g) + 2.0 moles NH 3 (E a 122.22 kJ/mol, n 1.41), respectively. The final residue is CoO + 6C. Thermal decomposition pathway is represented in Scheme 2 [32].  3(g) , respectively, leaving AgCo 2 O 5 + C residue. These steps showed small different thermal stability and T max . DTG peaks showed increasing ∆H due to Ag 0 NPs interaction with OH − of proline forming a robust self-assembled monolayer on Ag 0 NPs via strong Ag O covalent bond and Van der Waals interaction. The bond energy Ag-O is 217 kJ mol −1 . There is a small difference in ∆H confirmed supramolecular structure [33], see Scheme 3. 210, 276 and 320 °C, respectively, wt.loss 5.24 and 1.70% due to loss 4.0 coordinated H2O molecules. Fourth and fifth thermal decomposition processes Wt.loss 12.04 and 22.66% due to release 2H2O + C7H6 and C13H14 + 2NH3(g), respectively, leaving AgCo2O5 + C residue. These steps showed small different thermal stability and Tmax. DTG peaks showed increasing ΔH due to Ag 0 NPs interaction with OHof proline forming a robust self-assembled monolayer on Ag 0 NPs via strong Ag O covalent bond and Van der Waals interaction. The bond energy Ag-O is 217 kJ mol −1 . There is a small difference in ΔH confirmed supramolecular structure [33], see Scheme 3. Scheme 3. Thermal degradation of PCCAg 0 NPs. Co(II) ion is a covalently linked proline through nitrogen or oxygen atom. Doping proline CoCl2 crystal by Ag 0 NPs gives thermally stable self-assembled crystals. Ag 0 NPs is an inorganic linker. Thermal parameters and activation parameters, Ea, ΔS # (J K −1 mol −1 ), ΔH # (kJ K −1 mol −1 ) are collected in Table 4. Scheme 3. Thermal degradation of PCCAg 0 NPs. Co(II) ion is a covalently linked proline through nitrogen or oxygen atom. Doping proline CoCl 2 crystal by Ag 0 NPs gives thermally stable selfassembled crystals. Ag 0 NPs is an inorganic linker. Thermal parameters and activation parameters, Ea, ∆S # (J K −1 mol −1 ), ∆H # (kJ K −1 mol −1 ) are collected in Table 4.
By profile fitting and indexing the PXRD pattern, the crystal structure is solved using direct methods with simulated annealing implemented in software EXPO2014. The unit-cell parameters for PCC crystals are refined using Pawley/LeBail fit analysis [17,34]. PXRD data of PCCAg 0 NPs crystals are indexed with N-TREOR, and in Figure 6, the crystal lattice parameters are collected in Tables 5 and 6 including unit cell parameters and Rietveld refinements Rp, Rwp, and S 2.03 parameters are also described. By profile fitting and indexing the PXRD pattern, the crystal structure is solved using direct methods with simulated annealing implemented in software EXPO2014. The unitcell parameters for PCC crystals are refined using Pawley/LeBail fit analysis [17,34]. PXRD data of PCCAg 0 NPs crystals are indexed with N-TREOR, and in Figure 6, the crystal lattice parameters are collected in Tables 5 and 6 including unit cell parameters and Rietveld refinements Rp, Rwp, and S 2.03 parameters are also described.      Figure 7 showed the refined bond distance in PCC and PCCAg 0 NPs.    The crystal structure is shown in Figure 8. The crystal structure is shown in Figure 8.   Refinement PXRD patterns convert the approximate structure of PCCAg 0 NPs crystal into an actual structure, see Figure 10.    Refinement PXRD patterns convert the approximate structure of PCCAg 0 NPs crystal into an actual structure, see Figure 10.  [35]. The geometry around the Co(II) ion has the same chelating manner, bond length and bond angle as PCC formed distorted Oh, Ag 0 NPs bind Co and proline via Van der Waals interaction [36,37]. All bond lengths and bond angle • of PCC are collected in Table S2.  34 (2)° and 177.00 (3)°, respectively, are close to linearity. Average Co-O and Co-N bond lengths confirmed Co(II) distorted Oh geometry. Doped Ag 0 NPs had unchanged crystal geometry around the Co(II) ion type, but changed dimensions of the crystal size by expanding the crystal size [35]. The geometry around the Co(II) ion has the same chelating manner, bond length and bond angle as PCC formed distorted Oh, Ag 0 NPs bind Co and proline via Van der Waals interaction [36,37]. All bond lengths and bond angle° of PCC are collected in Table S2.
The room temperature polycrystalline X-band ESR spectral patterns Figure 11 showed a typical distorted axial pattern for high-spin Co(II) with a three resonances structure. The perpendicular signal is split into two components due to rhombic distortion.  The room temperature polycrystalline X-band ESR spectral patterns Figure 11 showed a typical distorted axial pattern for high-spin Co(II) with a three resonances structure. The perpendicular signal is split into two components due to rhombic distortion. Effective g values are g y 2.15, g x 1.98, and g z 1.89 for PCC and g y 2.12, g x 1.98 and g z 1.88 for PCCAg 0 NPs. Hyperfine coupling of electron spin with 59 Co nucleus (I 3/2 ) produces three equally spaced lines in the g y region with [A y 35 × 10 −4 cm −1 ] and in the g z region with [A z 45 × 10 −4 cm −1 ] corresponding to |−3/2> → |−1/2>, |+3/2> → |+1/2> and |−1/2> → |−1/2>. Splitting in the g x region is unobserved due to line-width. A x equals 15 × 10 −4 cm −1 ] [38][39][40].
Molecular orbital calculations, performed by qualitative analysis performed by Ab initio calculations following CASSCF method, Gaussian software program suggested distorted Oh symmetry around Co(II) ion. Elongated Co-O bonds with water molecules in comparison to proline favors magnetic anisotropy, (Figure 12) zero field splitting (ZFS) parameters D and E in Griffith Hamiltonian are obtained using five magnetic parameters: g x , g y , g z , D, and η following Equations (7) and (8), Bond length, Å and bond angle • of PCC are collected in Table S2 [41]: where parameters D xx , D yy , and D zz are principal values of ZFS tensor. Molecular orbital calculations, performed by qualitative analysis performed by Ab initio calculations following CASSCF method, Gaussian software program suggested distorted Oh symmetry around Co(II) ion. Elongated Co-O bonds with water molecules in comparison to proline favors magnetic anisotropy, (Figure 12) zero field splitting (ZFS) parameters D and E in Griffith Hamiltonian are obtained using five magnetic parameters: gx, gy, gz, D, and η following Equations (7) and (8) where parameters Dxx, Dyy, and Dzz are principal values of ZFS tensor. ZFS term causes splitting quartet ground state into two Kramers doublets with energy gap, Δ. ∆ = 2 1 + 3ɳ (4) where η = E/D is the rhombicity parameter. Table 7 showed a positive D value and relative high energy. Splitting between two Kramers doublets nevertheless remains larger than 130 cm −1 , a value that is within the characteristic range observed for other distorted Oh symmetry. Slow relaxation of single-molecule magnets (SMMs) arises from strong magnetic anisotropy, see Figure 12 and Tables 7 and 8 [42].
where η = E/D is the rhombicity parameter. Table 7 showed a positive D value and relative high energy. Splitting between two Kramers doublets nevertheless remains larger than 130 cm −1 , a value that is within the characteristic range observed for other distorted Oh symmetry. Slow relaxation of single-molecule magnets (SMMs) arises from strong magnetic anisotropy, see Figure 12 and Tables 7 and 8 [42]. Excitation energy, ∆, includes spin-orbit coupling effects [43]. Electrochemical behavior of crystals is represented in Figure 13. The AC conductivity of the sample is decreased with increasing frequency less than 10 5 Hz due to charges entrapped between grain boundaries and grains. The AC conductivity of crystals is enhanced by SNPs doping. High electric conductivity of samples enabled the applications as new thermoelectric materials for decreasing heat wastes for the The AC conductivity of the sample is decreased with increasing frequency less than 10 5 Hz due to charges entrapped between grain boundaries and grains. The AC conductivity of crystals is enhanced by SNPs doping. High electric conductivity of samples enabled the applications as new thermoelectric materials for decreasing heat wastes for the 4th generation of solar cells, new high-temperature superconductors and mini-magnets [44].
Proline showed no antimicrobial activity to any tested species. SNPs enhanced antibacterial activity of Co(II)-proline single crystals based on inhibition zones (IZ, mm), Table 9. Proline exhibited no inhibition effect on all tested microorganisms. PCC and PCCAg 0 NPs exhibited potent antibacterial and antifungal activities. High microbial activity of PCC crystals is due to dopant Ag 0 NPs. PCC and PCC Ag 0 NPs showed significant inhibition toward K. pneumoniae, E. faecalis, A. brasiliensis and C. Albicans in comparison to the reference standard ciprofloxacin antibiotic. Doping by Ag 0 NPs increased chelation complexation between the crystal and DNA of microbes [44].
Same mechanism of action leading antimicrobial and anticancer activity, metal complexes binding DNA of cancer cells.
High cytotoxic activity of PCCAg 0 NPs is due to Ag 0 NPs dopant increased DNA complexation. Cytotoxicity against the breast carcinoma cell line MCF-7 followed the order: PCCAg 0 NPs > PCC > proline. PCCAg 0 NPs exhibited over 5-fold less cytotoxicity against MRC-5 (IC 50 145.5 µM) than the reference cisplatin antitumor therapeutic drug. High toxicity of PCCAg 0 NPs against MRC-5 can be mitigated by using PCCAg 0 NPs in medication as a vial under the applied magnetic field to target tumor cells without affecting normal cells [45].
The dependence of cells viability, human breast adenocarcinoma (MCF-7) and human lung fibroblasts (healthy control) MRC-5, on crystal concentration confirmed that the SNPs crystal showed the best performance in terms of: highest toxicity to cancer cells and less toxicity on the normal lung cell lines approach effect of cisplatin [46][47][48]. All findings obtained in this current study confirmed that silver nanoparticles possess unique physicochemical characteristic-enabled applications in all field technologies [49,50].
complexation. Cytotoxicity against the breast carcinoma cell line MCF-7 followed the order: PCCAg 0 NPs > PCC > proline. PCCAg 0 NPs exhibited over 5-fold less cytotoxicity against MRC-5 (IC50 145.5 μM) than the reference cisplatin antitumor therapeutic drug. High toxicity of PCCAg 0 NPs against MRC-5 can be mitigated by using PCCAg 0 NPs in medication as a vial under the applied magnetic field to target tumor cells without affecting normal cells [45].   Higher values are observed for IC 50 of the tested samples for MRC-5 normal healthy lung cells: cisplatin (30.2) < PCCAg 0 NPs (145.5) < PCC (255.8). This trend showed that Ag 0 NPs improved antitumor activity of PCC crystal with low toxicity for normal lung cells.

Conclusions
New single crystals of proline amino acid doped by either cobalt chloride (CoCl 2 ) or CoCl 2 + AgNPs are prepared by an innovative low-cost approach (slow evaporation method at room temperature). AgNPs: successfully incorporated into proline single crystals; improved magnetic properties of showed good magnetic properties (µ eff. (B.M) for PCC from 4.44 B.M to 4.55 B.M.
Powder XRD diffraction patterns for PCC and PCCAg 0 NPs are obtained in good purity as a triclinic unit cell. Cobalt chloride created anisotropy magnetism in proline single crystals. PCCAg 0 NPs exhibited good antitumor activity against human breast adenocarcinoma (MCF-7). PCCAg 0 NPs crystal showed six-fold more cytotoxicity against breast cancer cell MCF-7, IC 50 22.1 µM, than the reference drug cisplatin, IC 50 11.7 µM. The mechanism of action of PCCAg0NPs could be binding and complexing DNA of cancer cells.
PCCAg 0 NPs showed five-fold lower cytotoxicity to normal healthy lung cells MRC-5, (IC 50 145.5 µM) than cisplatin (IC 50 30.2 µM). The high toxicity of PCCAg 0 NPs against MCF-7 and its low toxicity against MRC-5 makes it a promising candidate for chemotherapy of breast cancer.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines11020360/s1, Table S1: Assigned vibrational FTIR bands spectra of crystals, Table  S2: Bond length, Å and bond angle of PCC.  Data Availability Statement: All data in this study will be available on request.