Biosecurity Test of Conjugated Nanoparticles of Chitosan-Protoporphyrin IX-Vitamin B9 For Their Use in Photodynamic Therapy

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“Biosecurity Test of Conjugated Nanoparticles of Chitosanprotoporphyrin IX-

Vitamin B9 for Their Use in Photodynamic Therapy”
by Irving Trejo-Santillán, Citlali Cecilia Mendoza-Guevara, María del Pilar

Ramos-Godínez, Eva Ramón-Gallegos
published in the IEEE Transactions on NanoBioscience Early Access

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Transactions on NanoBioscience
Trejo-Santillán et al.: Biosecurity test of conjugated nanoparticles of chitosan-protoporphyrin IX-vitamin B9 for their use in photodynamic therapy.
Biosecurity test of conjugated nanoparticles of chitosan-

protoporphyrin IX-vitamin B9 for their use in photodynamic
therapy
Irving Trejo-Santillán, Citlali Cecilia Mendoza-Guevara, María del Pilar Ramos-Godínez, Eva
Ramón-Gallegos*
Abstract— The main obstacle of Photodynamic Therapy (PDT) (Ps), a light source (hv) of specific wavelength (lamps, laser, LED,
to damage and destroy abnormal cells is that most among others) and intracellular molecular oxygen (O2) to generate
photosensitizers (Ps) have a highly hydrophobic nature with a photodynamic reactions (PDR) leading to the death of target cells
tendency to aggregate in aqueous solutions and the non- through oxidative damage [2].
specificity towards target cells. Nanotechnology proposes new In order to PDT to be safe and effective it is crucial that Ps is
tactics for the development of monomeric Ps nanotransporters and
delivered in therapeutic concentrations to target cells (such as tumor
active targeting molecules with the use of biodegradable polymeric
cells), while at the same time it can be taken up in small amounts by
nanoparticles to improve the specificity towards target cells.
However, these products must comply with safety tests to be non-specific cells, and should be minimized undesirable side effects in
endorsed as therapeutic alternatives by regulatory organizations. healthy tissues [1]. However, there are two main obstacles to achieving
The goal of this work was to optimize the synthesis of chitosan this goal: 1) Most Ps have a π bond conjugation system, making the
polymeric nanoparticles conjugated with protoporphyrin IX and molecules flat and with a tendency to be highly hydrophobic, therefore,
vitamin B9 (CNPs-PpIX-B9) by the ionic gelation method from the they are prone to form aggregates in aqueous environments. This
established protocol previously carried out by our laboratory with aggregation decreases the efficiency of Ps, which must be in its
1.74 times fold of efficiency. They were characterized by ultraviolet monomeric form to be photoactive [1] [3] and 2) Ps generally do not
light-visible light, infrared spectroscopy and transmission electron bind specifically to tumor cells, making it difficult to target them only
microscopy. In addition, in CHO-K1 cells the biosafety (cytotoxicity to diseased tissue when PDT is applied [1]. To overcome these
and genotoxicity) of conjugate was assessed following the
obstacles, considerable efforts have been made to design
recommendations of the chromosomal aberrations test by OEDC
473 (2016) guideline. The conjugate did not show evidence of
administration systems that can incorporate Ps in its monomeric form
genotoxicity (clastogenicity). Surprisingly, the significant without diminishing its activity and without causing harmful effects.
differences between the treatments performed and the negative Many of these systems include applications of nanotechnology, which
control do not represent increases in chromosomal aberrations, has the approach of manufacturing nanomaterials (NMs), devices and
whereby the safe concentrations to use the conjugate without systems by manipulating matter on a scale of the order of one millionth
inducing cytotoxic or genotoxic effects are less than 0.25 mg / mL. of a meter. The active targeting of the drug by coupling multiple
Since it induced a significant decrease of structural chromosomal molecules on the surface of the NPs, such as antibodies, peptides,
aberrations, generating a positive effect on the genomic stability of aptamers, hyaluronic acid, folic acid (vitamin B9) and so on, avoid the
CHO-K1 cells cultured in this test system.
accumulation of Ps in nonspecific sites and improve its selective
interaction with target cells [1] [4]. Folic acid, also known as vitamin
Index Terms—Biosecurity, chromosomal aberrations,
B9 (B9), is essential for the proliferation, nucleotide biosynthesis and
cytotoxicity, genotoxicity, nanobiotechnology,
maintenance of some metabolic pathways of cells [5], making it one of
nanoparticles, nanotoxicity, photodynamic therapy.
the selected choices for anti-cancer therapy based on NPs directed at
tumor cells of epithelial origin, since they overexpress folate receptors
I. INTRODUCTION (FR) which favors the entry of NPs into the cell by receptor-mediated
Photodynamic therapy (PDT) is an alternative technological endocytosis [6] [7].
modality for the treatment of a variety of diseases that requires the In addition, drug-conjugated polymeric NPs offer numerous
destruction of pathogenic cells (e.g. cancer cells and infectious strategies to be manipulated by changes in their physicochemical
microorganisms) or the removal of unwanted tissue properties, such as molecular weight, biodegradability,
(neovascularization in the choroid and atherosclerotic plaques in the hydrophobicity, etc., with a variety of methods to efficiently
arteries) [1]. PDT is composed of three components: a photosensitizer encapsulate pharmaceutical molecules [4] [8]. Chitosan-based
polymeric NPs (CNPs) possess low toxicity, mucoadhesion,
This work was supported by Instituto Politécnico Nacional, grant SIP biocompatible, biodegradability and adsorption properties [9]. In
20180786 and SIP 20195320. addition, the Food and Drug Administration (FDA) approved chitosan
*Corresponding author: E. Ramón-Gallegos. for tissue engineering and drug delivery [10].
I. Trejo-Santillán, C. C. Mendoza-Guevara and E. Ramón-Gallegos
However, the formulation is required to be non-toxic and
(corresponding author), attached to the Escuela Nacional de Ciencias
biodegradable so that it is easily eliminated from the body by
Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Wilfrido
Massieu 399, Mexico. (e-mail: [email protected]; [email protected]; physiological mechanisms and to avoid any possibility of
[email protected], respectively). accumulation within cells that can generate cytotoxicity [11], due to
M. P. Ramos-Godínez is with the Laboratorio de Microscopía their small size, NPs can easily interact with biomolecules located both
Electrónica, Instituto Nacional de Cancerología (INCAN), Avenida San on the surface and inside cells [12].
Fernando 22, Mexico. (e-mail: [email protected]). Regulatory bodies such as the Organization for Economic
Cooperation and Development (OECD) and the International
1536-1241 (c) 2021 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
Conference on Harmonization (ICH) have published guidelines for a mortar to generate a finer powder and dissolved in a 0.5% v / v acetic
preclinical toxicity tests for chemical and pharmaceutical substances acid solution (Karal, SA of CV cat # 1000) at a concentration of 1 mg
[13]. Furthermore, the OECD has confirmed that the standard methods / mL with the help of a vortex shaker (optional, incubate at 35 ° C / 30-
for chemical compounds are applicable to NMs, but the risk 60 min / 120 rpm to better dissolve the chitosan solution). The previous
assessment is complicated by limited information on exposure to NMs solution was vacuum filtered with Whatman # 1 filter paper.
[14]. Since the exposure to NMs has increased dramatically in the last Subsequently, 25 mL of the chitosan solution were dissolved in 18 mL
century due to the rapid development of nanotechnology, of previously heated type I water and kept under magnetic stirring at
nanomedicine and the consumption of products containing NMs which 200 rpm. The pH was adjusted to 5.11 with 1 N NaOH solution. Then,
raises new health risks, therefore, the discipline in charge of evaluating 5 mL of pentabasic sodium triphosphate or pentasodium
the safety aspects of NMs is nanotoxicology [15]. tripolyphosphate (TPP) was added dropwise at 1mg / mL (Sigma-
The fact that NPs come into contact with cells, proteins, membranes, Aldrich, cat # 72061) to the chitosan solution dissolved in 0.5% acetic
DNA and organelles, establishes a series of nano-biointerfaces that acid and allowed to react under magnetic stirring at 700 rpm / 35 ° C /
depend on colloidal forces and dynamic biophysicochemical 15 min. Subsequently, the CNPs suspension was vacuum filtered with
interactions, which lead to the formation of particles surrounded by Whatman # 1 filter paper and dialyzed with a dialysis membrane
biomolecules, protein crowns, cell uptake and biocatalytic processes (Cellulose dialysis tubing benzoylated, 2000 MWCO. Sigma-Aldrich,
that could turn out to be biocompatible or bioadverse from the cat # D-7884) in double distilled water with stirring at 200 rpm / 24 h.
biosafety perspective of the use of NMs [16]. Therefore, genotoxicity
tests (GT) are used to determine if a pharmaceutical compound induces Conjugation of CNPs with PpIX and B9. To prepare 10 mL of the
genetic damage, which can cause a wide range of problems, including conjugate, 0.57 mL of N-hydroxysuccinimide (NHS) (MP
cancer and teratogenicity [17]. The evaluation of GT as an essential Biomedicals, cat # 02101880) at a concentration of 0.5 mg / mL and
safety test begins with a basic battery of in vitro tests followed by some 0.63 mL of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)
cases of in vivo tests [18]. Consequently, genotoxic substances can at a concentration of 0.5 mg / mL were mixed in an Erlenmeyer flask
induce changes from a single nucleotide (gene mutations), structural and incubated in a MaxQ 4450 incubator shaker (Thermo Fisher
(chromosomal mutations), and numerical chromosomal changes Scientific) at 200 rpm / 15 min. To the above mixture were added:
(genomic mutations) [19] as the most common changes in cancer cells 0.840 mL of disodium protoporphyrin IX solution (PpIX) at 1 mg / mL
[20]. In general, two classes of genotoxic agents are considered at the (Sigma-Aldrich, cat # P5889); 0.21 mL of folic acid at 1 mg / mL
chromosomal level, the clastogens and aneugens. The former cause (Sigma-Aldrich, cat # F8798) and 0.75 mL of type I water, keeping
structural chromosomal aberrations through two modes of action: 1) stirring at 200 rpm / 15 min in the dark. Subsequently, 7 mL of the
Direct (reactive to DNA), potentially triggering mutations through CNPs suspension were added and it was kept reacting under stirring at
chain breaks, replication of damaged DNA, inhibition of DNA 200 rpm / 2.5 h / 30 ° C in the dark. After the elapsed time, the previous
synthesis and errors in DNA repair, and 2) Indirect (non-reactive with suspension was centrifuged at 5000 rpm / 5 min (Eppendorf centrifuge
DNA) by the production of reactive oxygen species (ROS) or 5804R) and it was verified that the supernatant was homogeneous and
inhibition of DNA synthesis, which induce chromosome breakage [19] of a translucent red color. The resulting suspension was then dialyzed
[21]. On the other hand, aneugens cause numerical chromosomal with a dialysis membrane (Cellulose dyalisis tubing benzoylated, 2000
aberrations, generating genomic instability through the non-reactive MWCO. Sigma-Aldrich, cat # D-7884) in double distilled water with
mode of action with DNA (indirect), either due to stress on DNA magnetic stirring at 200 rpm / 23 h in the dark. Immediately afterwards,
replication or due to the inhibitory activity of the mitotic spindle [21] dialysis with icing sugar was continued for 1 h, also to concentrate the
[22]. Therefore, in this work it is proposed to optimize the synthesis of CNPs-PpIX-B9 conjugate, always covering the dialysis tube with a
the conjugate of chitosan-protoporphyrin IX-vitamin B9 NPs (NPQ – sufficient quantity and adding new icing sugar every 15 min. Finally,
PpIX – B9) previously obtained from [Gutiérrez-García and Ramón the CNPs-PpIX-B9 conjugate concentrate was centrifuged at 5000 rpm
Gallegos, 2018], as well as the tests of biosafety (cytotoxicity and / 5 min twice, recovering the supernatant and discarding the
genotoxicity) of this nanomaterial for its use in PDT. What we are precipitate.
looking for with the selection of this nanoconjugate is to ensure that
the application of this nanomaterial is safe during the development Characterization of the CNPs-PpIX-B9 conjugate by ultraviolet-
preclinical phase, so assessing the potential for toxicity of the visible (UV-Vis) spectroscopy. To confirm that the synthesis of the
nanomaterial is important to monitor the imminent risk and support its CNPs-PpIX-B9 conjugate was carried out, the characteristic
biosafety before proceeding to the next stage of development of the absorption spectrum of the conjugate in the wavelength range of 200
nanoconjugate and its later application in the PDT. to 800 nm was verified on a NanoDrop 2000 Spectrophotometer
(Thermo scientific).
II. METHODS
Characterization of the CNPs-PpIX-B9 conjugate by near infrared
The optimization of the synthesis of CNPs was carried out using the
(IR) spectroscopy. 1 mL of the conjugate was lyophilized and the
ionic gelation method and the conjugation of protoporphyrin IX
obtained powder was made into a tablet with potassium bromide
(PpIX) and B9 to the CNPs of the technique previously reported in our
(KBr), and the spectrum was acquired in a FT-IR Spectrometer
laboratory [23], based and modified from [24] and [25] to which
spectrophotometer (Spectrum 2000, Perkin Elmer).
additional modifications were made by changing parameters of the
reaction conditions, incubation, centrifugation and dialysis to optimize
Characterization of the CNPs-PpIX-B9 conjugate by transmission
the final product.
electron microscopy (TEM). One grid formvar / carbon coated - 200-
A. Synthesis, characterization and sterilization of the CNPs- mesh copper was negatively stained in 4% uranyl acetate for 20 min.
PpIX-B9 conjugate Finally, the grid was washed with type I water to remove excess
Chitosan nanoparticles synthesis (CNPs). Chitosan from the shrimp reagent and allowed to dry for 24 h. It was observed in a JEM-1010
shell ≥ 75% deacetylated (Sigma-Aldrich, Cat # C3646) was ground in transmission electron microscope (JOEL) at a power of 60 kV.
Sterilization, lyophilizing and storage of CNPs-PpIX-B9. To sterilize a) From the number of cells [31]
the CNPs-PpIX-B9, different methods were tested: 60Co gamma rays
with dose ranges (23.16 kGy to 25.75 kGy) at the National Institute for ln 2
𝑃𝐷𝑇 = 𝑡 ×
Nuclear Research (ININ), Mexico; pasteurization at 63-68 °C / 30 min 𝑋
ln .
in a water bath immediately changed to 4 °C and ultra-pasteurization 𝑋/
at 135-140 °C / 10 s / 2 cycles in oil bath by UV light / 15, 30 and 45 (2)
min and by autoclaving at 121 °C / 15 lb pressure / 20 min and by
filtration with a 0.22 µm pore diameter polyethersulfone (PES)
membrane (BIOFIL Syringe Filter). Then, aliquots were taken to mix Where t is the incubation time in any unit, Xb is the number of cells
with culture medium and incubated at 37 °C / 48 h to verify sterility. at the beginning of the incubation time and
Samples were also taken to verify the characteristic absorption Xe is the number of cells at the end of the incubation time.
spectrum (data not shown) of the conjugate before and after the b) From the slope (m) of the line
sterilization methods. The CNPs-PpIX-B9 conjugate was stored
refrigerated at 4 °C in the dark until use. log 2
A volume of 0.5 mL of CNPs-PpIX-B9 suspension was placed in a 𝑃𝐷𝑇 =
𝑚
FreeZone 4.5 L benchtop lyophilizer (Labconco) at minus 50 °C / < (3)
0.03 mbar / vacuum / 24 h and the obtained powder was weighed to
determine the concentration of the original suspension of the conjugate Where m is the slope and is defined as
in mg / mL.
log 𝑁 − log 𝑁'
B. Authentication and characterization of the CHO-K1 cell line 𝑚=
𝑡4 − 𝑡5
Cultivation and maintenance of the CHO-K1 cell line. The Chinese (3.1)
hamster ovary-K1 cell line (CHO-K1) were cultured and maintained
in Ham F-12 culture medium supplemented with 3.5% glucose and
10% fetal bovine serum (FBS) in 25 cm2 bottles (Corning Costar) Where N is the number of cells after t hours, N0 is the number of
incubated at 37 °C / 5% CO2 until cells reached 80-90% confluence initial cells, t2 is the elapsed time of N and t1 is the initial time of N0.
for subculturing, cell counting, authentication and characterization of To corroborate the result, the Doubling Time calculator online was
the cell line [26]. used [32].
Karyotype of the CHO-K1 cell line. Following the procedure of [33]
Cell morphology. The cells were observed directly under the briefly explained as follows: a quantity of 1x105 cells / mL was seeded
microscope frequently to identify and compare the state of the cells in duplicate in a 6-well microplate and incubated for 1 to 1.5 PD at 37
grown in low and high density in culture, without presenting any °C / 5% CO2. Three hours before completing the PD, a colchicine
unusual appearance [27]. solution was added to a final concentration of 0.25 µg / mL and it was
gently homogenized. Subsequently, the following steps were carried
Growth kinetics. From an 80-90% confluent subculture of CHO-K1 out:
cells, a cell suspension of 1 x105 cells / mL was adjusted. They were a) Cell harvest. At the end of the PD period, the cells were trypsinized
seeded in duplicate in 6-well microplates and each duplicate was to detach the cells from the plastic. After decanting the medium,
incubated at different times: 24, 48, 72, 96 and 120 h at 37 ° C / 5% the cell pellet was resuspended in the residual medium. Next, 5 mL
CO2. After the respective time of each duplicate, they were trypsinized of hypotonic KCl solution (J.T. Baker) at 75 mM were added and
to detach the cells from the plastic. For each of these cell suspensions, incubated for 10 min at room temperature.
the cell count was performed in duplicate at the corresponding b) Fixation of cells. 5 mL of cold Carnoy fixative solution (4 ° C)
incubation time. Subsequently, a semilogarithmic graph of the were added to the previous cell suspension and mixed gently. It
population growth curve of the CHO-K1 cell line was constructed. was centrifuged at 1500 rpm / 5 min. The supernatant was
Consequently, the population doubling (PD) was calculated with the discarded and resuspended again in 5 mL of cold Carnoy's solution
following expression [28] [29]: incubating ≥ 1 h or overnight at 4 °C. Subsequently, two washes
were carried out with 5 mL of cold Carnoy solution and
𝑁 centrifuging at 1500 rpm / 5 min in each wash. Then, the fixed cell
ln
𝑁' pellet was resuspended in 5 mL of the fixative solution to be stored
𝑃𝐷 =
ln 2 at 4 °C until use.
(1) c) Slice preparation. The cell suspension in Carnoy's solution was
centrifuged at 1500 rpm / 5 min and the supernatant was discarded.
Where, N is the number of cells after t hours, N0 is the number of Two or 3 drops of fixing solution were added with the help of a
initial cells. Pasteur pipette in the cell pellet to resuspend the upper part and
From the PD data, the population doubling time (TDP) was take a small volume of it. One drop of the cell suspension was
determined, calculated from the slope and taking as reference the linear dropped in about 30 cm in height onto clean, degreased slides
portion of the exponential growth phase of the curve. The logarithm of suspended in a 70 °C steam bath. They were allowed to air dry for
base 10 was applied to generate a straight line of the type y = mx + b. 24 h or 1 h at 60 °C.
In it, the independent variable x corresponds to the incubation time, d) Slide stain. They were stained with 10% Giemsa's solution (Hycel,
and the dependent variable y is the logarithm of the number of cells per cat # 6303) for 15 min. Next, washings were carried out with
unit area. The expressions to calculate the TDP by the slope of the line, running water and rinsed with double distilled water until excess
also called specific growth rate (k) [30] and by the number of cells, are dye was removed. The slides were allowed to air dry and mounted
indicated as follows: with synthetic resin-xylol (1: 1 v / v) and 24 x 50 mm coverslips.
A count of 100 cells in metaphase was performed to determine the B9 in each row of the microplate and incubated at 37 °C / 5% CO2 / 3
modal number of chromosomes and the construction of the karyotype h. In one row, only the vehicle was added as a token of viability. Next,
according to its morphological profile based on the chromosomal three washes with Dulbecco's phosphate buffer (D-PBS) were
rearrangements from [34]. performed in each well and fresh supplemented medium was added,
continuing the incubation until 1.5 PD was completed. The cells were
C. In vitro biosecurity assay (cytotoxicity) then harvested and the viable cell number counted as a method to
estimate cytotoxicity using the RPD and RICC values, as mentioned
According to the OEDC 473 (2016) guideline method [29], to above. As a positive cytotoxicity control, mitomycin C (MMC) was
identify substances that cause structural chromosomal aberrations; used in a concentration interval of 0, 0.025, 0.05, 0.1, 0.2 and 0.4 µg /
which can be caused by clastogenic events in mammalian cells grown mL, using the same RPD and RICC parameters.
in established cell lines due to their karyotype stability and the
frequency of aberrations spontaneous chromosomes; as well as the D. In vitro mammalian chromosome aberration test (OECD
considerations and limitations of the tests described in the guide, some 473) applied to CNPs-PpIX- B9
of them are briefly described next:
• The use of an exogenous source of metabolic activation is Following the recommendations of the OEDC 473 (2016) guideline,
required unless the cells are metabolically competent with respect genotoxicity bioassays (chromosomal aberrations) were performed in
to the test substance. In this case, we used 2% post-mitochondrial CHO-K1 cells with the following experimental conditions in
S9 fraction (induced by aroclor-1254 in Sprague Dawley male rats duplicated:
supplemented with cofactors mix from Moltox FT). a) Negative control (aqueous vehicle in which the conjugated
• Solvent do not exceed 10 % (v/v) in culture medium of final CNPs-PpIX-B9 was dissolved) and MMC as a clastogenic
treatment. positive control without metabolic activation in concentrations
• Evaluation at least three concentrations (no include positive of 0, 0.025, 0.05, 0.1, 0.2 and 0.4 µg / mL incubated by 3 h of
and negative controls) in appropriated cytotoxicity criteria. exposure, each to determine cytotoxicity.
• The highest concentration should try to reach 55 ± 5 % of b) Short (acute) exposure of CNPs-PpIX-B9 for 3 h at 0.5, 0.25 and
cytotoxicity or maximum solubility by means of the relative 0.125 mg / mL with metabolic activation (+ S9: 2% post-
population doubling values (RPD) and the relative increase in cell mitochondrial S9 fraction).
count (RICC) parameters (both consider the proportion of the cell c) Short (acute) exposure of CNPs-PpIX-B9 for 3 h at 0.5, 0.25 and
population that has been divided), it was calculated as follows: 0.125 mg / mL without metabolic activation (–S9).
Afterward, in the three previous cases, washes were carried out
Increase in number of cells in treated cultures and fresh culture medium was added, incubating until
(𝑓𝑖𝑛𝑎𝑙 − 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔) completing 1.5 PD.
𝑅𝐼𝐶𝐶 (%) = d) Long (chronic) exposure of CNPs-PpIX-B9 for 1.5 PD without
Increase in number of cells in control cultures
(𝑓𝑖𝑛𝑎𝑙 − 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔) washing, at concentrations of 0.5, 0.25 and 0.125 mg / mL
× 100 without metabolic activation (–S9).
(4)
E. Statistical analysis of the chromosomal aberration test
From previous treatments, 300 metaphases were counted with a

And number of centromeres equal to the modal number ± 2 per
concentration and controls, taking into account the frequency of
Number of 𝑃𝐷 in treated cultures chromatid-type and chromosome-type chromosomal aberrations,
𝑅𝑃𝐷 (%) = × 100 being equally divided between the replicates, if applicable, to conclude
Number of 𝑃𝐷 in control cultures
(5) as clearly negative or positive a test substance. Additionally, the
frequency of incidental aberrations (gaps, polyploidies, centromeric
interruptions, endoreduplication and others) was recorded separately,
Where PD is, the Population Doubling, and is defined as:
but not included in the frequency of total aberrations. The statistical
analysis was performed with the one-way Fisher's exact test,
𝑝𝑜𝑠𝑡 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟
log T 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 [ considering the percentage probability ≤ 1% (p ≤ 0.01) as significant,
𝑃𝐷 = calculated with the help of GraphPad Prism 5 software, version 5.01.
log 2
(5.1)
III. RESULTS
For example, a RICC, or a RPD of 53% indicates 47% cytotoxicity/ A. Synthesis, characterization and sterilization of the CNPs-
cytostasis.
PpIX-B9 conjugate
Cytotoxicity bioassays of CNPs-PpIX-B9 in CHO-K1 cells. A
The results of the optimization of the synthesis of the conjugated
quantity of 7x104 CHO-K1 cells / cm2 per well was seeded in a 24-well
CNPs-PpIX-B9 by the ionic gelation method using the UV-Vis
microplate (Corning Costar, cat # 3526) in duplicate and incubated at
absorption spectra shows a continuous spectrum with maximum
37 °C / 5% CO2 per 1 PD of cells. Next, a volume of supplemented
absorption peaks characteristic of each of the reactants (Fig. 1.), which
culture medium mixed with the filter-sterilized CNPs-PpIX-B9 was
suggests the formation of CNPs and the conjugation of PpIX and B9.
added, without exceeding 10% (v / v) of the aqueous solvent of the
The filter sterilization method (pore size 0.22 µm) of the unoptimized
CNPQ-PpIX-B9 conjugate in the culture medium. This to adjust to the
CNPs-PpIX-B9 conjugate was not adequate, because the characteristic
final concentrations of 0, 0.25, 0.5, 1, 2, and 4 mg / mL of CNPs-PpIX-
UV-Vis absorption spectrum was lost after filtering (Fig.2a).
Therefore, the other sterilization methods that were tested gave poor o performed according to their morphological profile, whereas it was
low results. However, it was observed that, in the previous methods, possible with Giemsa staining performing the karyotype analysis
contamination prevailed to a lesser or greater degree when the CNPs- according to the chromosomal rearrangements shown by [34] (Fig. 5.).
PpIX-B9 conjugate was incubated in culture medium for 48 h / 37 °C,
C. In vitro biosecurity assay (cytotoxicity)
except in filter sterilization, but the drop in the absorption spectrum
still imposed a huge drawback. Nevertheless, this made possible to
CNPs-PpIX-B9 cytotoxicity bioassays in CHO-K1 cells. The
determine that the CNPs-PpIX-B9 conjugate is autoclavable and has
determination of the maximum limit CNPs-PpIX-B9 cytotoxicity at 55
very thermostable structure.
± 5% using the recommended parameters of the RICC and RPD, is
On the other hand, since sterilization by filtration turned out to be
shown in Table I. Therefore, the conjugate generates a cytotoxicity
the best method, to overcome the disadvantage of the loss of the
with a supralinear dose-response curve (Fig. 6.), where the crossover
absorption spectrum of the CNPs-PpIX-B9 conjugate after filtration,
point of the percentage of viable and dead cells corresponds to 50% of
some conditions of the synthesis described in the Methods section were
cytotoxicity, with RICC values obtained of 59.25% and with a
modified, which were not included in the original synthesis. Therefore,
discrepancy of the RPD obtained of 77.69%. Both close to the
the changes allowed to increase the maximum absorption peaks of the
concentration of 0.5 mg / mL of the conjugate of CNPs-PpIX-B9 (see
characteristic spectrum of the conjugate (Fig. 2a.). Subsequently, filter
Table I and Fig. 6.), and both compared with the negative cytotoxicity
sterilization was carried out again and although the characteristic
control. For this purpose, the decrease of the RICC of 59.25% was
absorption spectrum was not completely lost, rather, it resembles the
chosen as which indicates a 40.75% cytotoxicity at the concentration
absorption spectrum before optimizing without filter sterilization (Fig.
of 0.5 mg of the conjugate of CNPs-PpIX-B9. Additionally, the effect
2a.), obtaining approximately 1.74 times of efficiency, in comparison
of the conjugate on the morphology of CHO-K1 cells in the tested dose
with the procedure previously performed in our laboratory.
range (0, 0.25, 0.5, 1, 2 and 4 mg / mL), which induced morphological
To confirm that the binding reactions were carried out after the
changes from 0.25 to 4 mg / mL, which probably correspond to cell
optimization of the CNPs and that there were no alterations in the
death due to apoptosis, necrosis or other, however, it remains to be
nanostructure, the IR spectrum turned out very similar with other IR
determined experimentally.
spectroscopy studies in the synthesis of NPQ by the ionotropic gelation
method [35], where the optimized CNPs-PpIX-B9 conjugate, the
Mitomycin C (MMC) cytotoxicity bioassays in CHO-K1 cells. The
characteristic peaks of some functional groups possessed by the
determination of the maximum limit MMC cytotoxicity at 55 ± 5%
nanostructure are observed. However, because it is a macromolecular
using the recommended parameters of the RICC and RPD is shown in
complex, some frequency bands of vibrational groups overlap in
Table II. For this purpose, the decrease in RPD of 59.2% was chosen,
certain intervals; such as groups of hydrogen bonding: N–H (from
which indicates a 40.8% cytotoxicity close to the dose of 0.1 µg / mL
amines and amides), O–H (from alcohols) and C–H (from methylenes)
of MMC. Therefore, MMC generates cytotoxicity with a dose-
in the range of 4000 cm-1 to 2850 cm-1; double bond groups: C=N
response curve with a supralinear tendency (Fig. 7.), where the
(from imines), C=O (from amide carbonyl groups) and C=C (from
crossover point of the percentage of viable and dead cells corresponds
ethylene groups) in the range of 2000 cm-1 to 1500 cm-1; and single
to 50% of cytotoxicity, with RICC values obtained of 45.7%. Close to
bond groups: C–O (from alcohols and ethers) and C–N (from amines)
the dose of 0.05 µg / mL of MMC and RPD obtained from 59.2%, close
in the range of the fingerprint region of 1500 cm-1 to 400 cm-1 (Fig. 2b
to the dose of 0.15 µg / mL of MMC (Table II and Fig. 7.), and both
and 2c.).
compared with the negative cytotoxicity control. Due to the differences
The CNPs-PpIX-B9 conjugate seen by TEM shows a morphological
difference before being optimized (Fig. 3a) and after being optimized in concentration, the average concentration between RICC and RPD of
(Fig. 3b). Before being optimized, CNPs-PpIX-B9 are observed with 0.1 µg / mL was considered. Additionally, no significant
heterogeneous shapes: spherical, hemispherical, rods, even serpentine. morphological changes induced by MMC were observed, except for
After optimization, the shape is more homogeneous: spherical and cell number reduction (data not shown).
hemispherical, so the average size before and after being optimized
changed, resulting in the frequency distribution graph of the conjugate D. In vitro mammalian chromosome aberration test (OECD
size (Fig. 3c), with an average 19.92 nm ± 7.52 nm. 473) applied to the CNPs-PpIX-B9 conjugate
B. Authentication and characterization of the CHO-K1 cell line
Table III shows the tabulated and summarized results of the
chromosome aberration test and their related values (incidental
Morphology cell and growth kinetic. CHO-K1 cell line has an observations) at three dose levels of the CNPs-PpIX-B9 conjugate,
adherent epithelial-like morphology and the growth kinetics. Fig. 4a. where the highest dose (0.5 mg / mL) corresponds to 55 ± 5%
shows the population growth curve with a characteristic sigmoid
cytotoxicity (RICC obtained 59.25%). Considering the aberrant
pattern composed of three phases: a lag or latency phase, a log or metaphases described above, the counts were performed for at least
exponential phase, and a stationary or plateau phase. Additionally, 300 metaphases per concentrations and control, and the incidental
determination of the Population Doubling Time (PDT) of the CHO-K1
aberrations were excluded from calculation of the aberrant metaphases
cells during the log phase, an exponential growth was obtained up to
percentage. Therefore, the CNPs-PpIX-B9 conjugate did not show
72 h of incubation so the calculated PDT resulted in 19.56 h (Fig. 4b.). evidence of genotoxicity (clastogenicity), but did show a significant
The same result was obtained using the Doubling Time calculator percentage decrease in chromosome aberrations at concentrations less
online [32]. than or equal to 0.25 mg / mL, when compared to the negative control;
whereas the control positive (MMC 0.1 µg / mL) presented significant
Karyotype of the CHO-K1 cell line. The modal number of increases in the incidence of metaphases with chromosomal
chromosomes of the cell line in question turned out to be 20, with some aberrations that confirm the sensitivity of the test at adequate toxicity
derivatives of 18, 19, 21 and 22, which reveals the hypodiploid levels in the absence and presence of metabolic activation (S9 mix) in
characteristic of the CHO-K1 cell line. Furthermore, since no CHO-K1 cells of this test system performed. In addition, Fig. 8, show
chromosomal banding were made, the order of the chromosomes was
some examples of chromosomal aberrations observed and considered
or analysis with simplified abbreviations used by the International entire region of aliphatic C–H bonds [37] [38] [39]. This also helps to
System for Human Cytogenetic Nomenclature (ISCN) [36]. verify the depletion of unconjugated carboxyl groups from PpIX and
B9 not covalently attached to the CNPs.
TABLE I On other hand, the size of NPs is comparable to the size of globular
CNPS-PPIX-B9-INDUCED RELATIVE CYTOTOXICITY INDICATORS IN proteins (2-10 nm), the diameter of the DNA helix (2 nm), and the
CHO-K1 CELLS. thickness of the cell membrane (10 nm), allowing them to easily enter
CNPs- RPD Cytotoxicity RICC Cytotoxicity cells and organelles. For example, NPs up to 50 nm in diameter appear
PpIX-B9
(%) (%) (%) (%) to enter cells and organelles (nucleus and mitochondria) by passive
(mg/mL)
4 -207.8 100 -17.01 100
diffusion [40]. Likewise, it is presumable that chitosan can also be
2 -150.3 100 -16.36 100
transported by transcytosis and paracellularly through tight junctions
1 6.28 93.7 2.21 97.7
of epithelia [10]. Therefore, it is likely that 19.92 nm ± 7.52 nm CNPs-
0.5 77.6 22.3 59.2 40.75
PpIX-B9 manage to enter the cytoplasm of CHO-K1 cells through
0.25 95.3 4.61 90.1 9.9
some receptor-mediated mechanism such as: folate receptor (FR),
Control 100 0 100 0
heme transporter protein 1 (HCP1) which really is a proton-coupled
folate transporter (PCFT) [41] [42] or even through endocytosis
RPD = relative population doubling, RICC = relative increase mediated by hemoglobin scavengers and heme group. Since the latter
in cell count
only differs from the PpIX in containing an iron II atom [43].
TABLE II Therefore, CNPs-PpIX-B9 conjugate-induced cytotoxicity is
MITOMYCIN C-INDUCED RELATIVE CYTOTOXICITY INDICATORS IN determined by the differences in the physicochemical properties,
CHO-K1 CELLS. whereby affect the biological-toxicological activity, mainly in three
MMC RPD Cytotoxicity RICC Cytotoxicity factors: surface characteristics (charge, area, reactivity and surface
(μg / mL) (%) (%) (%) (%) chemistry), particle size and entrance portal, as well as their
0.4 -14.1 100 -7.14 100 biopersistence [17] [44]. In the case of the charge surface
0.2 37.4 62.6 25.3 74.7 characteristics of NPs they have a strong tendency to form
0.1 59.2 40.8 45.7 54.3 agglomerates (held together by the outer surfaces by weak van der
0.05 61.3 38.7 48.05 51.9 Waals forces) and / or aggregates (held together or fused by strong
0.025 100 0% 100 0 chemical bonds) in the dry or suspended state, which determines the
Control 100 0% 100 0 real size (hydrodynamic diameter) of the NPs separated from each
other.
RPD = relative population doubling, RICC = relative increase in
cell count
Besides, in TEM images it can be observed that the optimization of
the synthesis of CNPs-PpIX-B9 conjugate shows an improvement
IV. DISCUSSION morphology and size of the nanoconjugate, which could be reflected
in the decrease of the hydrodynamic diameter. In addition, the
Optimization of the conjugated CNPs-PpIX-B9 showed a electrostatic stabilization can be controlled by variation of the
continuous spectrum in UV-Vis spectroscopy with maximum chemical environment, such as pH, which we modify in various
absorption peaks characteristic of each of the reactants (Fig. 1), occasion to optimized the nanoconjugate, providing in part, a probable
whereas infrared (IR) spectroscopy suggests that the binding reactions explanation of the polyelectrolyte surface of CNPs-PpIX-B9 conjugate
were carried out for the construction of the CNPs and the conjugation with a synergetic effect of electrostatic and steric stabilization. Due to
of PpIX and B9. their pH sensitivity (charge by protonation or deprotonation), the
Fig. 2b shows the stretch or elongation band for the O–H and N–H charge contribution to particle stabilization would become highly
bonds found between 3300 cm-1 and 3700 cm-1. That is, the band is dependent on the solution environment [58] [59].
probably relatively intense (wide) due to the overlap of the O–H bonds All these factors could partially explain the difficulty that arose at
(from hydroxyl of chitosan and B9, as well as the formation of the beginning before the optimizing synthesis of CNPs-PpIX-B9
hydrogen bonds) with the N–H bonds (from 2° amines, since that are conjugate. That is, if the unoptimized conjugate formed large amounts
seen as a single band, otherwise, the presence of 1° amines would be of aggregates–agglomerates that constituted a kind of network, due to
observed as a band with two peaks). Therefore, it suggests that it is impurities or defects generated during synthesis and the conjugation
from an amide bond that should have been formed when conjugating process, which resulted in a larger hydrodynamic diameter and / or
the carboxyl groups (-COOH) of PpIX and B9 with the -NH2 group close to the micrometric scale, with the consequent retention of the
from chitosan. Other characteristic bands from stretching vibration is conjugate in the PES membrane pore 0.22 µm, for which the loss of
the carbonyl group, which generates an intense peak between 1640 the characteristic spectrum in the UV-Vis region was observed (Fig.
cm-1 to 1820 cm-1 [37] [38]. However, to distinguish carbonyl from an 2a.). In contrast, the optimization synthesis of conjugate could have
aldehyde, ketone, carboxylic acid, ester or amide, it is necessary to eliminated some important proportion of impurities or defects,
know the position of the carbonyl band and that of other bands and whereby could allow the formation of fewer aggregates and / or
even other spectroscopic techniques, such as nuclear magnetic aggregates-agglomerates with a decrease in the hydrodynamic
resonance (NMR) [38] [39]. In this case, for 1° and 2° amides, the band diameter, thus achieving, filtration of the conjugate, which is
obtained in Fig. 2b. is between 1700 cm-1 to 1625 cm-1, in addition to consistent with the moderate loss of absorption intensity in the
knowing that there are C–N and N–H bonds, it can be inferred that the spectrum after optimization (Fig. 2a).
band of the carbonyl group corresponds to amide groups, which According to the average diameter of 19.92 nm ± 7.52 nm of CNPs-
suggests that this bond would correspond to the conjugation of PpIX PpIX-B9 conjugate gives them a high surface-volume ratio in
and B9 to CNPs. Another possible confirmation is the C=O bond, so it suspension, which is why it is responsible for the increase in surface
may correspond to an amide group and the absence of the characteristic reactivity and catalytic activity, so that the number of exposed surface
O–H bond absorption band from carboxylic acids, which is very molecules increases sharply exposing surface defects, void sites and
pronounced starting near 3330 cm-1 and extending through over the
chemical bonds that increase the chemical and oxide-reduction (redox) B9 or PpIX, thus also the cytotoxicity induced by the conjugate at high
reactivity of the nanoconjugate [15] [17]. The high positive surface doses of 1,2 and 4 mg / mL, may be due to the effect of the zeta
charge of the CNPs-PpIX-B9 conjugate could easily allow its entry potential of CNPs, since the degree of deacetylation of chitosan
into cells due to the electrostatic attraction between the negatively represents a relationship directly proportional to cytotoxicity, due to
charged cell membrane glycoproteins and the positively charged of the increase in positive charges, particularly of the amine groups in the
nanoconjugate due to the amine groups from B9 and PpIX, including CNPs surface [10] [60] [61][62].
chitosan, that is, from amine groups that have not been conjugated with
TABLE III
SUMMARY RESULTS OBTAINED IN THE IN VITRO CHROMOSOME ABERRATION TEST IN MAMMALIAN CHO-K1 CELLS.
Aberrant Number of aberrations Incidental observations a
Conc metaphases
Treatment Cht type Chr type
(μg/mL) O chtg chrg Pb Cb E
% p chtb chte chrb chre
Negative and Positive Controls
Water – 13 – 16 0 20 3 0 68 18 22 12 0
MMC 0.1 36 < 0.0001 * 43 8 39 10 8 83 63 33 11 0
Short exposure without metabolic activation (–S9)
125 3.3 < 0.0001 * 0 0 6 4 0 8 8 20 2 0

CNPs-PpIX-B9 c 250 4.6 0.0002 * 2 0 10 2 0 30 4 26 4 0
500 7.3 0.0150 4 0 14 4 0 30 6 28 6 0
Short exposure with metabolic activation (+S9)
125 18 0.057 22 0 26 4 2 46 12 18 12 0
CNPs-PpIX-B9 c 250 17.3 0.0859 18 0 30 2 0 66 22 26 8 0
500 16 0.1768 14 0 30 4 0 66 10 30 16 0
Long exposure without metabolic activation (–S9)
125 9.3 0.0973 4 0 18 6 0 38 12 16 6 0
CNPs-PpIX-B9 c 250 6.6 0.0065 * 10 0 8 0 2 32 4 34 8 0
500 6 0.0025 * 4 0 8 4 2 42 8 40 2 0
The p-value is the percentage probability of the one-way Fisher's exact test, where ≤ 1% (p ≤ 0.01) is considered
significant, indicated with an asterisk.
a chtg, chrg, P, C and E are excluded from the percentage calculation aberrant metaphases.
b There are no significant increases in the number of polyploid or endoreduplicating cells indicating potential for inhibition
of mitosis / aneugenicity or for inhibition of cell cycle progress, respectively.

c Slight reddish color change of the culture medium and no pH changes at the end of the treatment period, which is
consistent with the appearance of the CNPs-PpIX-B9 conjugate.

MMC= mitomycin C, cht= chromatid, chr= chromosome, chtb= chromatid breakage, chrb= chromosome breakage, chte=
chromatid exchange, chre= chromosome exchange, O= others include rings, pulverized chromosomes, and metaphases
with multiple aberrations greater than 5, P= polyploidy, C= centromeric interruption, E= endoreduplication.
In terms of biological response for the fields of pharmacology and nucleus of CHO-K1 cells and interacting with karyoplasmic DNA and
toxicology, particle sedimentation, diffusion and agglomeration as proteins, can generate primary genotoxicity, and if it is through the
related to their size, density, and surface properties strongly affect the generation of ROS, the mechanisms of primary genotoxicity would be
dose-response concept [58]. Also, the surface reactivity characteristics indirect, which may be due to the surface properties of CNPs-PpIX-B9
and their high surface-volume ratio of nanostructures expose surface when interacting with the negative charges of the DNA of the cells
defects, void sites, and chemical bonds that increase chemical and CHO-K1, which alters cell division, causes transcription disturbance
oxide-reduction (redox) reactivity, which can result in release of free and DNA damage, accelerating mutagenesis processes [60] [63].
radicals and reactive oxygen species (ROS) [15] [17]. This generates Moreover, there is evidence that NPs can deplete antioxidant stores,
oxidative stress, whereby leads to progressive cellular responses that increasing the potential for endogenous ROS and free radical levels to
can be classified as antioxidant defense, pro-inflammatory effects, and cause oxidative DNA damage [48]. However, according to the results
cytotoxicity [45] [46]. Considering all the above, toxic cell death obtained in Table IV, there is no genotoxic effect, if it is that there is
probably occurred because of the abrupt saturation of cellular defense interaction of the CNPs-PpIX-B9 conjugate with DNA, which needs
mechanisms with extraordinarily high doses at 1, 2, and 4 mg / mL of to be verified experimentally. However, genotoxicity does not need to
the CNPs-PpIX-B9 conjugate, which did not provide CHO-K1 cells be accompanied by mutagenesis or cytotoxicity, nor must cytotoxicity
with enough time to develop adaptive antioxidant responses [47]. necessarily be caused by genotoxic effects [64]. Likewise, the absence
Therefore, is why it was convenient to determine the maximum of genotoxicity (clastogenicity), denoted as a significant decrease in
concentration to test at 55 ± 5% cytotoxicity, as suggested OECD 473. structural chromosomal aberrations induced by CNPs-PpIX-B9 at
Furthermore, the average diameter of the CNPs-PpIX-B9 conjugate, concentrations less than 0.25 mg / mL, in comparison to the negative
according to measurements, is probably capable of translocating to the control, the positive effect of chromosomal aberrations reduction in
CHO-K1 cells CNPs-PpIX-B9 conjugate-induced can probably be I. Trejo-Santillán was a CONACyT grand holder (grant # 609251)
explained by the presence of the conjugated vitamin B9 (folic acid), during the present work. E. Ramón-Gallegos is COFAA, EDI and SNI
since mammals are not able to synthesize B9 de novo, it is obtained grant fellow.
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Fig. 1. Characteristic absorption spectrum of the CNPs-PpIX-B9 conjugate and the reactants for their synthesis. Absorption spectra correspond to
the ultraviolet-visible (UV-Vis) range. The approximate maximum absorption peak of each of the reactants is shown in parentheses. Abbreviations:
PpIX= protoporphyrin IX, B9= vitamin B9 (folic acid), C= chitosan; CNPs= chitosan nanoparticles, TPP= sodium triphosphate, EDC= 1-ethyl-3- (3-
dimethylaminopropyl), NHS= N-hydroxysuccinimide, CNPs-PpIX-B9= chitosan nanoparticles-protoporphyrin IX-vitamin B9 conjugate.
Fig. 2. (a) UV-Vis absorption spectra of the CNPs-PpIX-B9 conjugate optimizing their synthesis conditions and the effect of filter sterilization. The
optimization of the synthesis of the CNPs-PpIX-B9 conjugate showed an improvement by generally increasing the maximum absorption peaks in
the characteristic spectrum of the conjugate, and its decrease after filtration resembles to the intact non-optimized absorption spectrum, which
represents 1.74 times of efficiency, in comparison with the procedure previously performed in our laboratory. (b) CNPs-PpIX-B9 infrared spectrum
in the optimization of their synthesis conditions. The characteristic bands of some functional groups labeled with their wave number range, the type
of vibration: stretching (str) or bending (ben) are shown. However, due to the macromolecular structure of the conjugate of CNPs-PpIX-B9, the
superposition of the bonds of the groups of atoms is observed, which are labeled in the position most likely to be located. (c) Representation of the
expected amide bonds in the attachment of PpIX and B9 to the chitosan chains through their carboxyl and amine groups, respectively.
Fig. 3. Characterization of the size of CNPs-PpIX-B9. a) Photomicrographs obtained by TEM before optimizing and b) after optimizing. c) Histogram
and frequency polygon of the diameter distribution of the CNPs-PpIX-B9. The average diameter is 19.92 nm ± 7.52 nm. The bar is 100 nm.
Fig. 4. Population growth kinetics of the CHO-K1 cell line. (A) Growth curve with semilogarithmic representation of cell density versus incubation
time with a characteristic S-shaped sigmoid pattern that includes lag, log and plateau phase. The calculated population doubling time (PDT) is
indicated with dotted lines. The length of the bars at each point indicates the standard error. (B) Representation of the linear function of the correlation
between the logarithmic cell density growing exponentially during the incubation time and the lapses of the PD are indicated as calculated during
the kinetics.
Fig. 5. Karyotype representative of modal numbers of chromosomes of CHO-K1 cell line. (a) Karyotypic schematization of the CHO-K1 cell line and
cells in primary culture (lung) of chinese hamster based on results with BAC-FISH (bacterial artificial chromosome in situ fluorescent hybridization).
CHO-K1 regions homologous to chinese hamster primary cells are colored according to the diploid chromosomes of chinese hamster primary cells.
Taken and adapted from [34]. (b) Representative karyotypes obtained from the modal number (mn) of chromosomes of the CHO-K1 line (19, 20, 21
and 22) stained with 10% Giemsa. The results are based on the morphological profile from (a). 1000 X magnification.
Fig. 6. Cytotoxic effect of the CNPs-PpIX-B9 conjugate on the CHO-K1 cell line. The dose-response curve generates a crossover point of the viable
and dead cells percentage that corresponds to 50% cytotoxicity. The values of 55 ± 5% RICC and RPD are closer to the concentration of 0.5 mg /
mL of CNPs-PpIX-B9 (59.25% RICC obtained and with a discrepancy of 77.69% RPD obtained), which means 40.75% and 22.3% cytotoxicity,
respectively.
Fig. 7. Cytotoxic effect of MMC on the CHO-K1 cell line. The dose-response curve generates a crossover point of the viable and dead cells
percentage that corresponds to 50% cytotoxicity. Although in this case, 55 ± 5% RICC is closer to the concentration of 0.05 μg / mL of MMC (45.7%
RICC obtained), which indicates a 54.3% cytotoxicity, whereas the 59.2% RPD cytotoxicity obtained It is found at a concentration of 0.1 μg / mL of
MMC, which indicates a 40.8% cytotoxicity.
Fig. 8. Example of chromatid-type and chromosome-type aberrations of CHO-K1 cells considered for analysis. Aberrations are indicated with
arrowheads and with simplified abbreviations used by the ISCN: chtg= chromatid gap, chte= chromatid exchange (tr, triradial, qr, quadriradial),
chtb=chromatid breakage; chrg= chromosome gap, chrb= chromosome breakage (a= acentric, fis= centric fission, dup-fis= centric fission with
duplication of the kinetochore), chre= chromosome exchange (dic, dicentric; add (q? )= material of unknown origin added to the long arm of the
chromosome). In case of incidental-type aberrations of CHO-K1 cells, this were not considered within count for analysis: disp cen= centromeric
disruption, polyp <2n> + = ploidy level with chromosome gain (polyploid) with numerical abnormality in relation to modal number (mn 18-22) almost
diploid (2n), r= ring chromosome, pvz chr= powdered chromosomes [36]. 1000 X magnification.

Biosecurity Test of Conjugated Nanoparticles of Chitosan-Protoporphyrin IX-Vitamin B9 For Their Use in Photodynamic Therapy

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IEEE Xplore ®

“Biosecurity Test of Conjugated Nanoparticles of Chitosanprotoporphyrin IX-

by Irving Trejo-Santillán, Citlali Cecilia Mendoza-Guevara, María del Pilar

published in the IEEE Transactions on NanoBioscience Early Access

Biosecurity test of conjugated nanoparticles of chitosan-

From previous treatments, 300 metaphases were counted with a

Negative and Positive Controls

125 3.3 < 0.0001 * 0 0 6 4 0 8 8 20 2 0

of mitosis / aneugenicity or for inhibition of cell cycle progress, respectively.

consistent with the appearance of the CNPs-PpIX-B9 conjugate.

laboratory diagnostics and precision medicine,” J Lab Precis Med., vol.

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