Small

Electrostatically Assembled Nanoparticles for Effective Chemoimmunotherapy

--Manuscript Draft--

Manuscript Number: smll.201502230

Full Title: Electrostatically Assembled Nanoparticles for Effective Chemoimmunotherapy

Article Type: Full Paper

Section/Category:

Keywords: Caspases, cell cycle arrest, mitochondrial disruption, tumor associated macrophages,
Th1 immune response

Corresponding Author: Manish K. Chourasia, Ph.D.

Central Drug Research Institute
LUCKNOW, uttar pradesh INDIA

Additional Information:

Question Response

Please submit a plain text version of your July 27, 2015

cover letter here.
If you are submitting a revision of your
manuscript, please do not overwrite your
original cover letter. There is an
opportunity for you to provide your
responses to the reviewers later; please
do not add them here.
To
The Editor in Chief
Small
Dear Dr. José Oliveira
I am enclosing herewith manuscript entitled “Electrostatically Assembled Nanoparticles
for Effective Chemoimmunotherapy” for publication in small. The current manuscript
deals with development and optimization of electrostatically assembled doxorubicin
loaded immunotherapeutic nanoparticles. These nanoparticles have both chemo and
immunotherapeutic properties and can therefore facilitate chemoimmunotherapy single
handedly. With the submission of this manuscript I would like to undertake that the
manuscript has not been published elsewhere, accepted for publication elsewhere or
under editorial review for publication. I look forward to hearing from you.

Thank you
Manish K. Chourasia
Sr. Scientist
Pharmaceutics Division,
CSIR-Central Drug Research Institute,
Lucknow, UP, India 226031.
Tel: +91 2772450, 2772550
FAX: +91 2771941, 2771942
E-mail: manish_chourasia@cdri.res.in

Corresponding Author Secondary

Information:

Corresponding Author's Institution: Central Drug Research Institute

Corresponding Author's Secondary

Institution:

First Author: vivek Pawar

First Author Secondary Information:

Order of Authors: vivek Pawar

yuvraj singh

komal sharma

arpita Srivastav

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
 Jaya Meher

Pankaj Singh

Abhisheak Sharma

Akhilesh Singh

Himangshu Bora

Dipak Datta

Jawahar Lal

Manish K. Chourasia, Ph.D.

Order of Authors Secondary Information:

Abstract: Immunotherapeutic nanoparticles (NPs) could be a viable option for delivering

circumstantially effective cytotoxic agents in a manner which suppresses their toxic
manifestations. In light of the above hypothesis, doxorubicin (DOX) loaded NPs were
prepared using fucoidan (FCD), an immunomodulatory polysaccharide. FCD NPs
offered improved cytotoxicity (2.64 folds), cell cycle arrest in G1-S phase (34.65%) and
apoptosis (66.12 %) in tumor cells compared to free DOX. The enhancement in
apoptosis was due to raised mitochondrial depolarization (88.00 %). In vivo anticancer
activity in 4T1 induced tumor bearing BALB/c mice demonstrated a 2.95 folds
enhanced efficacy of the NPs after 26 days of treatment. Importantly, NPs treatment
generated an immunotherapeutic response indicated by gradual increment of the
plasma IL-12 levels and reversed polarization of tumor associated macrophages
(TAMs) towards M1 subtype. Furthermore, pharmacokinetic study suggested that NPs
administration in tumor infested mice caused serum DOX levels to vary in a biphasic
pattern, with twin peaks occurring at 1 h and 6h which help in maintaining preferential
drug localization in tumor. Acute toxicity study exhibited improved safety of DOX when
packaged as NPs. Conclusively; developed NPs would be an excellent approach for
improved immune-chemotherapy (in terms of efficacy, safety and immunocompetency)
against cancer.

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Complete Manuscript

Electrostatically Assembled Nanoparticles for Effective Chemoimmunotherapy

1 Vivek K. Pawara e, Yuvraj Singha e, Komal Sharmaa e, Arpita shrivastava, Jaya Gopal Mehera, Pankaj
2 Singha e, Abhisheak Sharmab e, Akhilesh Singhc, Himangshu K. Borad, Dipak Dattac, Jawahar Lalb,
3
4
Manish K. Chourasiaa e*
5
a
6 Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow-226031, U.P., India
7 b
Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-
8
9
226031, U.P., India
c
10 Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow-226001, U.P., India
d
11 Laboratory Animals Facility, CSIR-Central Drug Research Institute, Lucknow-226031, U.P., India
12 e
Academy of Scientific & Innovative Research, New Delhi-110025, India
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48 *Corresponding author
49 Manish K. Chourasia
50 Pharmaceutics Division,
51 CSIR-Central Drug Research Institute,
52
53
Lucknow-226031, U.P., India
54 Tel: +91 2772450, 2772550
55 FAX: +91 2771941, 2771942
56 E-mail: manish_chourasia@cdri.res.in
57
58
59
60
61
62 1
63
64
65
 ABSTRACT

1 Immunotherapeutic nanoparticles (NPs) could be a viable option for delivering circumstantially

2
3
4
effective cytotoxic agents in a manner which suppresses their toxic manifestations. In light of the
5
6 above hypothesis, doxorubicin (DOX) loaded NPs were prepared using fucoidan (FCD), an
7
8 immunomodulatory polysaccharide. FCD NPs offered improved cytotoxicity (2.64 folds), cell cycle
9
10
11 arrest in G1-S phase (34.65%) and apoptosis (66.12 %) in tumor cells compared to free DOX. The
12
13 enhancement in apoptosis was due to raised mitochondrial depolarization (88.00 %). In vivo
14
15
16 anticancer activity in 4T1 induced tumor bearing BALB/c mice demonstrated a 2.95 folds enhanced
17
18 efficacy of the NPs after 26 days of treatment. Importantly, NPs treatment generated an
19
20
21 immunotherapeutic response indicated by gradual increment of the plasma IL-12 levels and reversed
22
23 polarization of tumor associated macrophages (TAMs) towards M1 subtype. Furthermore,
24
25
pharmacokinetic study suggested that NPs administration in tumor infested mice caused serum
26
27
28 DOX levels to vary in a biphasic pattern, with twin peaks occurring at 1 h and 6h which help in
29
30 maintaining preferential drug localization in tumor. Acute toxicity study exhibited improved safety
31
32
33 of DOX when packaged as NPs. Conclusively; developed NPs would be an excellent approach for
34
35 improved immune-chemotherapy (in terms of efficacy, safety and immunocompetency) against
36
37
38 cancer.
39
40 Keywords: Caspases, cell cycle arrest, mitochondrial disruption, tumor associated macrophages,
41
42
43
Th1 immune response
44
45 1. Introduction
46
47 Balance of homoeostatic machinery is the most important regulator of human well-being. Even
48
49
50 under exposition of the harshest insults, homeostasis attempts to preserve the structural and
51
52 functional integrity, i.e. the body actively tries to cure itself. An immune response can be purported
53
54
55 as an illustrative example, wherein the homeostatic machinery strains to restore the basal functional
56
57 level of the body. The most fascinating feature of any immune response, regardless of scope and
58
59
60 context of the stimulant, is its incessant ubiquitousness, which tends to offer more pros than cons.
61
62 2
63
64
65
 However there are times in which an unwarranted or unintended immune response jeopardizes the

1 entire pretext for which the response was instigated in the first place, case in point being the
2
3
4
pathologic progression of breast cancer. Immunoreactivity towards breast carcinoma cells is
5
6 expressed by lymphoid infiltration into the tumor environment and regional lymphatic alterations,
7
8 which initially inhibit but later due to cross-talk between immunologic representatives (specifically
9
10
11 the phenotypic subtypes of tumor associated macrophages: M1 and M2) starts promoting cancer
12
13 propagation. It is therefore no wonder that scientists have tried to target this complex immunologic
14
15
16 interplay, to provide immunotherapy of breast cancer.[1, 2] Unfortunately though, any clinical
17
18 implication is still far-fetched, as stand-alone immune modulation has been shown to be only
19
20
21 capable of priming the body to fight tumor, and not actually eradicate it under in vivo conditions.[3,
22
23 4]
24
25
The next plausible evasive maneuver in clinically tackling breast cancer therefore seems
26
27
28 adaptation of a combinatorial strategy amalgamating the circumstantially effective chemotherapy
29
30 with a facilitatory immunomodulative regimen to obtain a focused counter measure free from
31
32
33 incidences of adverse effects or subpar efficacy.[5, 6] The reason of current work is same to deliver
34
35 anti-cancer drug via a stable, hydrophilic, biodegradable and non-toxic immunotherapeutic
36
37
38 platform.[7] In our previous study, we have stated an example of single particle based
39
40 chemoimmunotherapy via a nanoemulsion platform[8].
41
42
43
FCD, a sulfated polysaccharide, is extracted from the cell wall matrix of the brown algae for
44
45 example Fucus vesiculosus, Ecklonia kurome, Laminaria angustata, Ascophyllum nodosum, and
46
47 Hizikia fusiforme. Out of several well established biological properties,[9] FCD has welcome
48
49
50 immunomodulatory[10] and anticancer activity[11] and its usage to construct a drug delivery system
51
52 for killing cancer cells is conveniently expected to augment the cytotoxic action of the ferried drug
53
54
55 molecule.
56
57 DOX, an anthracycline antibiotic, is essential component of breast cancer chemotherapeutic
58
59
60 regimen and consequently has been selected here to provide cytotoxic thrust to the envisaged
61
62 3
63
64
65
 immunotherapeutic system.[12, 13] Chemotherapy with DOX, however, is associated with cardiac

1 toxicity,[14] forcing researchers to come up with alternate delivery strategies such as its inclusion in
2
3
4
carriers such as NPs.[15, 16] We have consequently followed suit and developed electrostatically
5
6 assembled NPs of DOX via polyelectrolyte complexation method (PEC) which utilizes
7
8 intermolecular electrostatic interactions offered by oppositely charged polymers, namely cationic
9
10
11 polyethylenimine (PEI) and anionic FCD. NPs have recently come to the fore with several
12
13 nanomedicine based products gaining clinical approval and there is room for many more if we
14
15
16 consider some serendipitous offerings they provide in targeting cancer.[17, 18] Due to their
17
18 miniscule size, NPs furnish a novel pattern of chemotherapeutic delivery in cancer cells by
19
20
21 presenting a maximal payload directly to intracellular locations like nucleus. They do so by
22
23 modulating the binding capacity and penetrability of cell membrane and different cellular
24
25
organelles. NPs also inadvertently accumulate in fenestrated tumor microenvironment (EPR effect)
26
27
28 paving the way for preferential tumoral localization of chemotherapeutic agents thereby avoiding
29
30 nonspecific toxicity.[19-21] Finally NPs can mold the inherent pharmacokinetic properties of the
31
32
33 drug they carry by controlling its presentation to systemic dispositional forces, thereby catalyzing
34
35 the drug’s efficacy.[22]
36
37
38 This manuscript details the pharmaceutics, pharmacokinetic, biologic, toxicologic and
39
40 deliberations expended in establishing the premier objective of the current study: development of
41
42
43
DOX bearing nanoparticulate system with immunotherapeutic credentials for treatment of breast
44
45 cancer.
46
47 2.0 Results
48
49
50 2.1 Synthesis of NPs and physicochemical characterization
51
52 Electrostatically assembled NPs were successfully prepared via PEC. It proved to be a
53
54
55 straight forward technique for development of stable NPs in ambient conditions. NPs were
56
57 effectively formed due to interpolyelectrolyte complexation between cationic PEI and anionic FCD.
58
59
60 Synthesis of blank NPs (PEI-FCD NPs) was confirmed chemically and physically through FTIR
61
62 4
63
64
65
 spectroscopy and TGA. Figure 1A and B represents the functional groups involved in establishment

1 of ionic bonds for development of NPs and morphological characteristics of the NPs investigated
2
3
4
through transmission electron microscopy (TEM) which depicted spherical structure of NPs.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23 Figure 1 (A) Polyelectrolyte complexation reaction scheme for synthesis of PEI-FCD NPs (B)
24
25 TEM photomicrographs depicting the morphological characteristics of the PEI-FCD-DOX
26
27
28 NPs.
29
30 Amino groups of PEI (3412.56 cm-1), carbonyl (1671.35 cm-1) and sulphate groups (1384.8
31
32
33 cm-1; S=O asymmetric stretching and 813.45 cm-1; C-O-S stretching) of FCD play major role in the
34
35 synthesis of PEI-FCD NPs (Figure S1A, Supplementary information). These peaks could be
36
37
38
employed to detect any ionic interaction between functional groups of PEI and FCD to synthesized
39
40 PEI-FCD NPs. The FTIR spectra of PEI-FCD NPs showed slight shifting of above mentioned bands
41
42 from 3412.56 to 3416.1 cm-1 (amino stretching of PEI), 1671.35 to 1679.65 cm-1 (carbonyl
43
44
45 stretching of FCD), 1384.8 to 1388.59 cm-1 (S=O stretching of FCD) and 813.45 to 817.28 cm-1 (C-
46
47 O-S stretching of FCD). These red shifts in the characteristic peaks of PEI and FCD might be due to
48
49
50 ionic interaction between representative functional groups. Further, TGA curves of PEI-FCD NPs
51
52 (Figure S1B, Supplementary information) suggested that NPs have different physical behavior than
53
54
55 individual formative components, PEI and FCD. PEI and FCD showed two (110 and 315 °C) and
56
57 three degradation stages (207, 325 and 437 °C) in their respective TGA curves whereas PEI-FCD
58
59 NPs had three different degradation stages (221, 520 and 710 °C). These alterations in degradation
60
61
62 5
63
64
65
 stages might be occurred due to physical metamorphosis of PEI and FCD. The ionic interaction

1 between characteristic functional groups of PEI and FCD along with their physical metamorphosis
2
3
4
presented adequate evidence for us to purport successful synthesis of PEI-FCD NPs.
5
6 The effect of weight ratio of PEI and FCD (1:0.25, 1:0.50, 1:1, 1:2 and 1:3 w/w) on the
7
8 physicochemical parameters such as average hydrodynamic diameter (DH), polydispersity index
9
10
11 (PDI), zeta potential and entrapment efficiency of the NPs (PEI-FCD NPs and PEI-FCD-DOX NPs)
12
13 was evaluated. The concentration of PEI was kept constant during the experimentation. The particle
14
15
16 size of the NPs was noticed in range between 41.1±17.39 to 159.2±18.34 nm. Raising the
17
18 concentration of FCD up to a certain level significantly increased zeta potential from -33.7±1.03 to -
19
20
21 45.1±0.49 mV beyond which (PEI:FCD weight ratio 1:3 or more) it stagnated and bulky
22
23 macroscopic precipitation was noticeable. The DH, PDI and zeta potential of blank NPs (PEI-FCD
24
25
NPs) was observed 64.9±07.03 nm, 0.153±0.042 and -44.2±0.35mV, respectively. The PDI data
26
27
28 suggested that NPs were uniformly dispersed.
29
30 Electrostatically assembled NPs offered excellent entrapment efficiency for DOX. DOX
31
32
33 encapsulation efficiency in NPs also improved significantly from 34.87±8.34 to 85.66±4.30% when
34
35 concentration of the FCD was increased. The ratio 1:2 for PEI:FCD weight was considered as
36
37
38 optimized ratio after that it provided high entrapment efficiency (82.20±2.50%), without
39
40 compromising the size (<100 nm) or zeta potential (-42.1±0.49 mV) of the NPs.
41
42
43
In vitro drug release from PEI-FCD-DOX NPs was observed at different pH to reveal effect
44
45 of pH on release of drug trapped inside NPs. The results demonstrated that DOX release varied
46
47 considerably in two different tested pH conditions as depicted in Figure S2, Supporting information.
48
49
50 DOX released at a faster rate in pH 5.5 than pH 7.4. The dissolution plot also displayed an initial
51
52 burst release of drug followed by a steadier more controlled pattern. About 24 and 29 % DOX was
53
54
55 released from the NPs at pH 7.4 and pH 5.5, respectively within first 24 h followed by a sustained
56
57 release pattern with almost 36 and 44 % DOX release respectively at pH 7.4 and pH 5.5 after 240 h.
58
59
60
61
62 6
63
64
65
 PEI-FCD NPs and PEI-FCD-DOX NPs were dispersed in culture medium to assess their

1 stability. Variation in particle size and zeta potential, with respect to passage of time was monitored
2
3
4
as the statistical marker of stability. Encouragingly, even after incubating PEI-FCD NPs and PEI-
5
6 FCD-DOX NPs in the culture medium for 36 h, DH and zeta potential showed insignificant change
7
8 in comparison to the readings obtained initially (Figure S3A and B, Supporting information). The
9
10
11 purpose of this simplistic assay, which was to establish the unmodified existence of developed
12
13 formulations in the culture media whilst conducting various other assays, was well and truly
14
15
16 established.
17
18 2.2 NPs exhibited excellent in-vitro antiproliferative activity
19
20
21 The in vitro antiproliferative activity of free DOX, PEI-FCD NPs and PEI-FCD-DOX NPs
22
23 in terms of IC50 was calculated on the basis of MTT assay. To calculate these, percent cell death was
24
25
plotted against the corresponding log concentrations of drug employed in each test and nonlinear
26
27
28 regression was performed. The variation in IC50 values with respect to different incubation periods
29
30 of free DOX, PEI-FCD NPs and PEI-FCD-DOX NPs with MDA-MB-231 cells and 4T1 cells are
31
32
33 summarized in Table 1 and Table S1, Supporting information.
34
35 These results suggested that developed PEI-FCD-DOX NPs were 2.65 times (MDA-MB-231
36
37
38 cells) and 2.73 (4T1 cells) more efficacious than free DOX after 72 h treatment. Moreover, DOX
39
40 loaded NPs were also found to be 12.80 times (MDA-MB-231 cells) and 6.72 (4T1 cells) more
41
42
43
effective than blank PEI-FCD NPs. The IC50 exhibited by free DOX, PEI-FCD NPs and PEI-FCD-
44
45 DOX NPs differed significantly [P<0.001 (n=3)] with passage of time i.e. after 48 and 72 h in both
46
47 the cell lines. However, there was insignificant difference between IC50 values of free DOX and
48
49
50 PEI-FCD-DOX NPs after 24 h in MDA-MB-231 cells and significant difference at level **P<0.01
51
52 (n=3) between IC50 values of free DOX and PEI-FCD-DOX NPs in 4T1 cells. Cytotoxic potential of
53
54
55 PEI-FCD-DOX NPs against MDA-MB-231 cells and 4T1 cells was higher compared to free DOX.
56
57
58
59
60
61
62 7
63
64
65
 Table 1 Representative IC50 values obtained after incubating free DOX, PEI-FCD NPs and

1 PEI-FCD-DOX NPs with MDA-MB-231 cells for 24, 48 and 72 h

2
3
4 IC50 values (µM)
Time (h)
5 Free DOX PEI-FCD-DOX NPs PEI-FCD NPs
6 24# 0.1492±0.039 0.1770±0.092 0.5568±0.139
7 48 ### 0.1135±0.085 0.0641±0.023 0.4940±0.062
8
9
72### 0.0954±0.034 0.0359±0.019 0.4597±0.056
#
10 Non-significant difference between PEI-FCD-DOX NPs versus free DOX and significant
11 difference between PEI-FCD-DOX NPs and PEI-FCD NPs at level P<0.05.
12 ###
Significant difference at level p<0.001 between PEI-FCD-DOX NPs versus free DOX and PEI-
13 FCD-DOX NPs versus PEI-FCD NPs.
14
15
16 2.3 PEI-FCD-DOX NPs arrest tumor cells in G1-S phase and induced apoptosis
17
18 In order to ascertain the molecular mechanism behind the in-vitro anti-proliferative activity of NPs,
19
20
21 cell cycle arrest study, apoptosis assay, measurement of mitochondrial potential and caspase activity
22
23 studies were performed. In our studies we found, NPs were found to elicit a programmed cell death
24
25
26 by restricting the cell cycle progression in G1-S phase followed by cellular apoptosis. Figure 2A
27
28 shows the results of cell cycle analysis obtained after incubating free DOX, PEI-FCD NPs and PEI-
29
30
31
FCD-DOX NPs with MDA-MB-231 cells for varying time intervals. Cells were initially arrested in
32
33 G1-S phase when treated with free DOX and PEI-FCD-DOX NPs followed by subsequent
34
35 increment in sub-G0/G1 phase population suggesting an increase in hypodiploid cellular
36
37
38 demographic which was indicative of cells undergoing apoptosis. Also PEI-FCD-DOX NPs (35.93
39
40 to 64.97%) induced greater cell cycle arrest in G1-S phase in comparison to free DOX (32.74 to
41
42
43 57.08%) in the 36 h window, probably due to sustained and specific release of DOX in the cytosol.
44
45 Following the same trend, the sub-G0/G1 phase population for PEI-FCD-DOX NPs (34.65%) was
46
47
48
also on a higher side after its exposition for 36 h compared to free DOX (8.05%). Even the blank,
49
50 PEI-FCD NPs, were found to arrest cells in G1-S phase (58.97% at 36 h) and raised sub-G0/G1
51
52 phase population (10.75% at 36 h). In 4T1 cells, PEI-FCD-DOX NPs exhibited the highest cell
53
54
55 cycle arrests in G1-S phase (70.00%) followed by PEI-FCD NPs (68.50%) and free DOX (62.82%)
56
57 after 36 h treatment (Figure S4, Supporting information).
58
59
60
61
62 8
63
64
65
 Annexin V-FITC and propidium iodide dependent assay was executed to assess the early

1 and late apoptotic cell population after treating MDA-MB-231 cells with free DOX, PEI-FCD NPs
2
3
4
and PEI-FCD-DOX NPs for varying time intervals. Early and late apoptosis is shown respectively
5
6 by the cells accumulating in lower right and upper right quadrant of the Annexin V-FITC and
7
8 propidium iodide dot plot. Results demonstrated that all the tested groups caused time dependent
9
10
11 enhancement in apoptosis, however inter-individual variation in the extent of apoptosis was also
12
13 found (Figure 2B). After 36 h of treatment, free DOX induced early and late apoptosis in 11.42 and
14
15
16 0.14% cells respectively. The respective percentage of cellular population in early and late apoptotic
17
18 phase was 66.12 % and 0.30% in groups treated with PEI-FCD-DOX NPs. PEI-FCD NPs also
19
20
21 caused 21.84% of cells to undergo early apoptosis and 7.07% cells to experience late apoptosis after
22
23 36 h. Treatment of 4T1 cells (36 h) with NPs also demonstrated enhancement in percent apoptotic
24
25
cells compared to PEI-FCD NPs and free DOX (Figure S5, Supporting information). Fluorescent
26
27
28 microscopic images of MDA-MB-231 cells treated with NPs depicted modified nuclear morphology
29
30 due to DNA fragmentation or chromatin condensation (Figure S6, Supporting information).
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 9
63
64
65
 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48 Figure 2 (A) MDA-MB-231 cell cycle distribution obtained after treating cells with free DOX,
49
50
51 PEI-FCD NPs and PEI-FCD-DOX NPs for different time intervals. Cells were stained with
52
53 propidium iodide and quantitative estimation was performed on a flow cytometer.
54
55
56 Distribution of cells in subG0/G1 phase, G2-M phase and G1-S phase is represented
57
58 individually in each cell cycle. Higher numbers of cells were arrested in G1-S phase after
59
60
treatment with PEI-FCD-DOX NPs compared to free DOX and PEI-FCD NPs, in 36 h. The
61
62 10
63
64
65
 simultaneous increment in the subG0/G1 population suggests induction of apoptosis. (B)

1 Apoptotic activity expressed by free DOX, PEI-FCD NPs and PEI-FCD-DOX NPs after
2
3
4
treating MDA-MB-231 cells for different time intervals. A propidium iodide and FITC-
5
6 Annexin V based dual staining assay was performed to assess the early and late apoptotic cell
7
8 population. Dot plot contain four quadrants to demonstrate the early (lower right), late (upper
9
10
11 right) and necrotic (upper left) cell population.
12
13
14
15
16 2.4 PEI-FCD-DOX NPs disrupted mitochondrial membrane potential and induce caspase
17
18 dependent apoptosis in tumor cells
19
20
21 Alteration in mitochondrial membrane potential of MDA-MB-231 cells after treatment with free
22
23 DOX, PEI-FCD NPs and PEI-FCD-DOX NPs was assessed in order to appraise molecular pathway
24
25
behind their apoptotic activity. JC-1 (fluorescent cationic dye) was utilized to measure disruption of
26
27
28 mitochondrial membrane potential. It emits fluorescence at 590 nm in healthy cells after binding to
29
30 mitochondria, and on the other hand it emits fluorescence at 525 nm in apoptotic cells due to
31
32
33 dispersed accumulation in entire cytosol. Results supported that free DOX, PEI-FCD NPs and PEI-
34
35 FCD-DOX NPs significantly interrupted the mitochondrial membrane potential in a time dependent
36
37
38 manner (Figure 3A). PEI-FCD-DOX NPs (88.00%) displayed higher mitochondrial depolarization
39
40 in comparison to free DOX (26.08%). Additionally, PEI-FCD NPs (66.91%) also disrupted the
41
42
43
mitochondrial membrane, most probably due to presence of FCD. Similar trend was followed when
44
45 4T1 cells were treated with NPs. The mitochondrial disruption activity of NPs in 4T1 cells was
46
47 higher compare to free DOX and PEI-FCD NPs (Figure S7, Supporting information). These results
48
49
50 were confirmed by fluorescence microscopy. Fluorescence photomicrographs represented that NPs
51
52 treatment retarded the red fluorescence intensity (appearing as yellow fluorescence in merged
53
54
55 images) suggesting accumulation of JC-1 in the cytosol due to disruption of mitochondrial potential
56
57 of the cells (Figure 3B).
58
59
60
61
62 11
63
64
65
 Caspases belong to class of cysteine proteases and are subsequently grouped into different classes

1 based on their sequence homology. Some caspases are known to work as initiator/signaling caspases
2
3
4
(caspases -2, -8 and -9) whereas others are recognized as effectors such as caspases -3, and -6. The
5
6 kinetics of caspase activation was assessed using caspase fluorogenic substrates. Caspases -3 and -9
7
8 were activated after treatment with free DOX, PEI-FCD NPs and PEI-FCD-DOX NPs. Activation
9
10
11 of caspase-9 was substantially high compared to caspase -2 and -8 indicating capsase -9 might act as
12
13 initiator caspase for intrinsic apoptotic pathway. Relatively low levels of caspases -6 were observed
14
15
16 suggesting capsase -3 may be the effecter caspase for intrinsic apoptotic pathway (Figure S8A,
17
18 Supporting information). The preferential activation of caspase -3 and -9 sealed the mechanism of
19
20
21 apoptosis induced by NPs to be intrinsic.
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 12
63
64
65
 Figure 3 (A) Mitochondrial depolarization activity represented by free DOX, PEI-FCD NPs

1 and PEI-FCD-DOX NPs after incubating with MDA-MB-231 cells up to different time
2
3
4
intervals. Mitochondrial membrane potential disruption was quantified using fluorescent
5
6 cationic dye JC-1. Dot plot shows two distinct population of cells displayed in lower
7
8 (depolarized cells) and upper (polarized cells) quadrant. (B) Fluorescence photomicrographs
9
10
11 displayed accumulation of non-aggregated JC-1 dye in cytosol (increased green fluorescence
12
13 intensity) upon treatment with NPs indicating depolarization of mitochondrial membrane.
14
15
16
17
18 2.5 PEI-FCD-DOX NPs modulate differentiation of J774.1A derived macrophages
19
20
21 J774.1A macrophages and 4T1 cells were co-cultured in presence of CoCl2 up to 24 h which
22
23 provided hypoxic environment to recruit M2 phenotype of macrophages. Further, NPs were
24
25
incubated with cells in fresh medium (without CoCl2). Effect of NPs treatment on differentiation of
26
27
28 macrophages was performed through M2 phenotype specific biomarkers i.e. CD206. FACS
29
30 histogram revealed that NPs treatment decreased the CD206 expression in NPs treated cells (Figure
31
32
33 4A). Compare to control cells, percent population of M2 phenotype of J774.1A macrophages was
34
35 decreased 1.59 and 1.94 folds respectively when treatment was given with PEI-FCD NPs and PEI-
36
37
38 FCD-DOX NPs. Treatment of free DOX did not pursue the similar results. These results indicated
39
40 that in vivo administration of NPs would contribute to the modulation of TAMs differentiation in
41
42
43
order to facilitate an immune based tumor rejection.
44
45 2.6 PEI-FCD-DOX NPs enhanced secretion of Th1 cytokine
46
47 As NPs showed a modulating effect on macrophage differentiation thus it would be interesting to
48
49
50 observe whether NPs treatment effected the secretion of Th1 and Th2 immune cytokines to provide
51
52 an immune dependent anticancer activity. To study this, NPs were incubated with J774.1A
53
54
55 macrophages co-cultured with 4T1 cells. Results demonstrated that NPs significantly unregulated
56
57 Th1 immune response and in contrast, downregulated the Th2 immune response (Figure 4B).
58
59
60 Compare to control cells, secretion of IL-12 (Th1 immunity specific biochemical marker) was
61
62 13
63
64
65
 enhanced 2.02 and 2.34 folds upon treatment with PEI-FCD NPs and PEI-FCD-DOX NPs,

1 respectively whereas secretion of Th2 immunity specific biochemical marker IL-4 was found to be
2
3
4
decrease 2.40 and 2.71 folds. The treatment of DOX did not depict any immunotherapeutic effect.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Figure 4 Immunomodulatory effects of NPs through modulating J774.1A macrophages
39
40 differentiation and enhanced release of Th1 immune cytokine. NPs were incubated with
41
42 J774.1A macrophages previously co-cultured with 4T1 cells for 24 h. Phenotypic evaluation of
43
44
45 macrophages and cytokine estimation was performed through flow cytometry and ELISA
46
47 kits, respectively. (A) Representative FACS histogram demonstrating down-regulation of
48
49
50 CD206 expression or differentiation of macrophages towards M2 phenotypes as showed by
51
52 reduction in CD206 specific macrophages population. (B) NPs treatment facilitated an
53
54
55 immunotherapeutic response indicated by enhanced expression of IL-12 and reduced
56
57 expression of IL-4. Asterisk represented significant difference between normal saline
58
59
60
versus*p<0.05 and normal saline versus **p<0.01.
61
62 14
63
64
65
 1 2.7 PEI-FCD-DOX NPs suppressed 4T1 induced tumor growth in syngeneic mice
2
3
4
In vivo antitumor activity was explored in breast tumor bearing BALB/c mice after considering the
5
6 scope of in vitro results ascertained so far. The tumor was induced via 4T1 cells. Free DOX, PEI-
7
8 FCD NPs and PEI-FCD-DOX NPs were intravenously injected at dose equivalent to 4 mg/kg DOX
9
10
11 at various time points, with careful measurement of tumor volume throughout the study. Results
12
13 demonstrated that PEI-FCD-DOX NPs had significantly higher tumor inhibitory activity in
14
15
16 comparison to free DOX and PEI-FCD NPs (Figure 5 A and B). PEI-FCD-DOX NPs were 2.95
17
18 (P<0.01) and 4.53 (P<0.001) folds more efficacious than free DOX and PEI-FCD NPs, respectively.
19
20
21 Tumor burden was estimated after finishing the study and results depicted that PEI-FCD-DOX NPs
22
23 treated animals exhibited lowest tumor burden (Figure 5C). Tumor burden was found to be 9.57
24
25
(P<0.001), 3.26 (P<0.05), and 6.85 (P<0.001) fold lower in animals treated with PEI-FCD-DOX
26
27
28 NPs compared to control, free DOX and PEI-FCD NPs respectively.
29
30 2.8 PEI-FCD-DOX NPs reversed TAMs polarization to M1 subtype and enhanced IL-12 levels
31
32
33 TAMs are divided into two distinct subtypes i.e. M1 and M2. M1 macrophages are known as
34
35 classically activated macrophages and induce secretion of IL-12 whereas M2 macrophages termed
36
37
38 as alternatively activated macrophages induce secretion of IL-10 and IL-4. Modulation of TAMs
39
40 polarization could facilitate immune based tumor rejection and prevent tumor metastasis.[23] TAMs
41
42
43
differentiation into M2 type was assessed after treatment with developed NPs and results indicated a
44
45 suppression of M2 polarization. M2 type TAMs favors tumor progression through metastasis.
46
47 Inhibition of M1 to M2 polarization would subvert tumor growth by avoiding generation of
48
49
50 resistance which in turn would favor immune system mediated tumor rejection. The population of
51
52 M2 TAMs was detected after treatment with NPs using flowcytometry and it was conveniently
53
54
55 found to be reduced, suggesting inhibition of M2 polarization. Results demonstrated that M2 TAMs
56
57 population was 2.56 and 4.42 folds lower in comparison to control group when animals were treated
58
59
60 with PEI-FCD NPs and PEI-FCD-DOX NPs, respectively (Figure 5D).
61
62 15
63
64
65
 Immunomodulatory property of NPs was assessed in in vivo conditions employing tumor

1 bearing BALB/c model. Immunotherapeutic response evoked by developed NPs was ascertained by
2
3
4
quantifying secreted cytokine marker of M1 (IL-12) and M2 (IL-4) subtype in plasma using an
5
6 enzyme-linked immunosorbent assay. The results indicated that NPs induced activation of IL-12 in
7
8 treated animals. The profile of IL-12 expressed by treated and control animals are represented in
9
10
11 Figure 5E and its concentration was increased gradually when animals were treated with PEI-FCD-
12
13 DOX NPs and PEI-FCD NPs. Animals treated with PEI-FCD-DOX NPs evidenced 7.48 folds
14
15
16 (p<0.001) increase in plasma IL-12 levels after 26 days. Animals treated with PEI-FCD NPs
17
18 showed 7.70 folds (p<0.001) enhanced level of IL-12 while free DOX treated animals did not
19
20
21 exhibit significant enhancement in the IL-12 levels (p< 0.05) on 26th day of the study. On the other
22
23 hand, plasma IL-4 levels were found to be 3.27 and 1.69 folds lower when animals were treated
24
25
with PEI-FCD-DOX NPs and PEI-FCD NPs, respectively after 26th days of the study (Figure 5F).
26
27
28 All treated groups were compared with control group (normal saline).
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 16
63
64
65
 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36 Figure 5 (A and B) Tumor regression study representing the efficacy of the developed NPs.
37
38 Free DOX, PEI-FCD NPs and PEI-FCD-DOX NPs (4 mg/kg) were administered intravenously
39
40
41
on 10th, 14th, 18th and 22nd day of the study and tumor volume was noticed during the study.
42
43 Animals were sacrificed on last day of the study and tumors were harvested. Photograph
44
45 displays the morphology of tumors taken from different animal groups. Asterisks represents
46
47
48 the significant difference versus control group (**p<0.01 and ***p<0.001). (C) Tumor burden
49
50 measured after completion of study. Result depict that lowest tumor burden was carried by
51
52
53 animal group treated with PEI-FCD-DOX NPs. Asterisks symbolizes the significant difference
54
55 versus control group (*p<0.05 and ***p<0.001). (D) Effect of NPs treatment on TAMs
56
57
58 differentiation was noticed using CD206 marker. FACS histogram represented that TAMs
59
60 population was polarized towards M1 subtype suggesting induction of IL-12 secretion or
61
62 17
63
64
65
 classical activation of macrophages. (E and F) Estimation of plasma IL-12 and IL-4 levels in

1 the 4T1 cells induced tumor bearing BALB/c mice was done by employing enzyme-linked
2
3
4
immunosorbent assay. Blood samples were collected before dosing and on 26 th day of the
5
6 study. Developed NPs significantly enhanced the plasma IL-12 levels whereas free DOX did
7
8 not exercise any significant effect on the plasma IL-12 levels suggesting immunotherapeutic
9
10
11 effect might be exerted by FCD. On treatment with NPs, lower plasma IL-4 levels were
12
13 observed after 26th day indicating downregulation of Th-2 immune response. Asterisks
14
15
16 demonstrating the significant difference between **p<0.01 versus normal saline, ***p<0.001
17
18 versus normal saline, ###p<0.001 versus normal saline and ns depicted no significant difference
19
20
21 versus normal saline (p<0.05).
22
23
24
25
2.9 NPs modulate the pharmacokinetics & biodistribution of the DOX
26
27
28 A LC-MS/MS based validated bioanalytical method was employed for quantifying DOX in serum
29
30 and tissue homogenates. DOX was quantified using ion transitions m/z 544.2 to m/z 361.1.
31
32
33 Pharmacokinetics and biodistribution profile of DOX after loading into NPs was ascertained and
34
35 compared with free DOX in order to delineate the effect of carrier system. Figure 6A and B
36
37
38 represents comparative serum profile of free DOX and DOX loaded NPs after intravenous
39
40 administration into tumor bearing mice. The Cmax was observed at 0.083 (1129.80±325.37 ng/mL)
41
42
43
and 1 h (1480.26±434.85 ng/mL) respectively for free DOX and NPs. Non-compartmental analysis
44
45 was performed to compute different pharmacokinetic parameters (Table 2). Upon administration as
46
47 PEI-FCD-DOX NPs, t1/2 (h) and AUC0-∞ (h*ng/mL) of DOX was extended 1.30 fold (p<0.05) and
48
49
50 2.69 folds (p<0.001) compared to its native form. Furthermore, NPs reduced clearance (mL/h/kg)
51
52 and elimination rate constant (Ke; 1/h) of DOX by 2.69 (p<0.01) and 1.26 folds.
53
54
55 The biodistribution of free DOX and DOX loaded NPs was assessed in liver, spleen, kidney,
56
57 heart and tumor. DOX was extensively distributed in most organs just after intravenous
58
59
60 administration. In case of PEI-FCD-DOX NPs, the DOX concentration was found to be
61
62 18
63
64
65
 significantly higher (p<0.05) in spleen and liver. Encouragingly the same animal groups had

1 significantly lower (p<0.05) DOX levels in heart and kidney after 24 h, when compared to free
2
3
4
DOX administered mice. The most important manifestation of repackaging DOX as PEI-FCD-DOX
5
6 NPs was significant improvement (p<0.05) in tumor drug levels compared to free DOX.
7
8 Biodistribution study further revealed that levels of DOX in liver, spleen, kidney and heart declined
9
10
11 with passage of time (Figure 6C).
12
13 Table 2 Pharmacokinetic parameters observed after intravenous administration of free DOX
14
15
16 and developed NPs to BALB/c mice bearing tumor at a dose equivalent to 4 mg/kg DOX
17
18 Pharmacokinetic Parameters PEI-FCD-DOX NPs Free DOX
19
Cmax (ng/mL) 1480.26±434.85 1129.80± 325.37
20
21 Tmax (h) 1 0.083
22 t1/2 (h)* 37.20±4.61 28.57±2.03
23 AUC 0-∞ ( h*ng/mL)*** 7454.79±960.24 2767.10±672.68
24 Clearance (mL/h/kg)** 536.68±80.23 1445.55±240.25
25
26
Ke (1/h) 0.019±0.008 0.024±0.004
27 *p<0.05,**p<0.01,***p<0.001 level of significance difference between free DOX versus PEI-FCD-
28 DOX NPs
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45 Figure 6 Biodistribution profile of DOX after intravenous administration of free DOX and
46
47
48 PEI-FCD-DOX NPs at dose equivalent to 4 mg/kg BALB/c mice bearing 4T1 tumor. (A)
49
50 Serum drug profile at specified time intervals showing rapid clearance of DOX. (B) Inset
51
52
53 shows initial time points for improved reader reception. (C) Organ distribution profile (liver,
54
55 spleen, kidney, heart and tumor) demonstrating DOX concentration at various time points in
56
57
58
different organs. Data represents mean±s.d (n=3).
59
60
61
62 19
63
64
65
 2.10 NPs improved safety profile of DOX

1 To gauge toxicity aspects of developed NPs, change in body weight, serum biochemical markers
2
3
4
and histological analysis was performed after intravenous administration of normal saline, PEI-FCD
5
6 NPs and PEI-FCD-DOX NPs in healthy BALB/c mice. There was no significant (p<0.05) change in
7
8 body weight of the animals treated with PEI-FCD NPs and PEI-FCD-DOX NPs whereas animals
9
10
11 treated with free DOX showed significant (p<0.001) decline in body weight of animals during
12
13 dosing followed by recovery of body weight (Figure 7A). Moreover, animals treated with free DOX
14
15
16 demonstrated significant (p<0.05) enhancement in liver function enzymes such as aspartate
17
18 aminotransferase (AST) and alanine aminotransferase (ALT). Compared to control group,
19
20
21 cardiotoxicity markers including creatine kinase (CK) and lactate dehydrogenase (LDH) were also
22
23 found to be significantly (p<0.001) increased upon treatment with free DOX. In contrast, animals
24
25
treated with PEI-FCD NPs and PEI-FCD-DOX NPs did not show any significant (p<0.05)
26
27
28 modification in liver function enzymes and cardiotoxicity markers (Figure 7B).
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 20
63
64
65
 Figure 7 Assessment of modification in body weight (A) and serum biochemical marker (B) of

1 healthy animals upon treatment with NPs. Multiple equivalent doses (10 mg/kg) of the DOX
2
3
4
was administered to healthy mice through tail vain and blood samples were collected at the
5
6 end of the study (14th day). Animals receiving free DOX exhibited decline in body weights
7
8 compared to control group whereas animals treated with NPs did not show any significant
9
10
11 change in the body weight. Compared to control group, liver function enzymes (AST and
12
13 ALT) and cardiotoxicity markers (CK-MB and LDH) were elevated upon treatment with free
14
15
16 DOX. NPs prevented the adverse effect of the DOX and improved its safety profile.
17
18
19
20
21 Stained section of liver, spleen, heart, kidney and lung were observed under an optical
22
23 microscope to assess the toxic implications. Normal architecture is depicted in tissue section of
24
25
spleen and kidney after treatment with normal saline, free DOX, PEI-FCD NPs and PEI-FCD-DOX
26
27
28 NPs. Potential toxic reactions were detected in liver and heart sections of animals treated with free
29
30 DOX. However, normal architecture was noticed in liver and heart sections of animals treated with
31
32
33 PEI-FCD NPs and PEI-FCD-DOX NPs. Histological analysis of animals treated with free DOX
34
35 indicated signs of necrosis associated with capillary congestion and vascular changes in heart and
36
37
38 displayed hemorrhage and congestion in liver (Figure 8).
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 21
63
64
65
 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37 Figure 8 Acute toxicity study displaying histological analysis of different organs after treating
38
39 with control (normal saline), free DOX, PEI-FCD NPs and PEI-FCD-DOX NPs. Normal
40
41
42 architecture was observed in all the tissue sections treated with normal saline, PEI-FCD NPs
43
44 and PEI-FCD-DOX NPs whereas sign of toxicity were evident in heart (necrosis associated
45
46
47 with capillary congestion and vascular changes) and liver (hemorrhage and congestion)
48
49 sections of animals treated with free DOX.
50
51
3. Discussion
52
53
54 The current study demonstrates fabrication of FCD NPs including the molecular mechanism
55
56 behind its chemoimmunotherapeutic action. PEI, FCD and DOX containing electrostatically
57
58
59 assembled NPs (PEI-FCD-DOX NPs) were successfully developed by mild stirring at ambient
60
61 temperature. The nanometric size of the NPs indicated their suitability towards intravenous
62 22
63
64
65
 administration and passive targeting capabilities to tumors due to EPR effect.[24] During

1 optimization it was observed that augmentation in concentration of FCD, beyond a limit (using PEI:
2
3
4
FCD weight ratio 1:3 or above) led to rapid size growth of NPs. It is extremely plausible that
5
6 increased concentration of FCD yielded more charged entities leading to greater electrostatic
7
8 interaction between the amino group of PEI and sulphate group of FCD, which consequently leads
9
10
11 to precipitation of particles. The theory associating charge proportionality to raised concentration of
12
13 FCD was substantiated by constantly decreasing zeta potential measurements of formed NPs. The
14
15
16 negative zeta potential is representative of charge supplied by sulfate groups on FCD. Further, DOX
17
18 entrapment efficiency was found to be enhanced when concentration of FCD was increased. The
19
20
21 raised entrapment was even more profound in NPs developed using PEI:FCD in weight ratio 1:2,
22
23 mostly due to interaction of amino group present in the sugar moiety of DOX to the sulfate group of
24
25
FCD. Drug release studies showed that NPs possessed pH dependent drug releasing property,
26
27
28 especially accentuated at acidic pH, presenting a case for selective drug release in cytosolic
29
30 locations. The facilitation in drug release at acidic pH can be attributed to re-protonation of the
31
32
33 amino group available in the sugar moiety of DOX at lower pH resulting in improved drug
34
35 solubility of drug in dissolution media. The implications of this effect are multimodal. The distinct
36
37
38 release rates at differing pH sets up a solid background for simulating the dichotomous pH
39
40 differentiation between blood (pH 7.4) and cancerous sites (slightly acidic). pH sensitive release is
41
42
43
desirable as slower release of DOX in neutral environment such as plasma (pH 7.4) or regular non-
44
45 cancerous intracellular locations will reduce the chance of nonspecific toxicity; this in turn increases
46
47 the probability of attaining cytotoxic concentrations due to faster release inside cancer cells (slightly
48
49
50 acidic pH 5.5).[25-27]
51
52 The improved antiproliferative activity of PEI-FCD-DOX NPs at extended time period could
53
54
55 be assigned to sustained drug release pattern offered by the NPs which provides cytotoxic drug
56
57 concentration in the cells for elevated time periods. Even the dummy PEI-FCD NPs also exhibited
58
59
60 quantifiable antiproliferative activity possibly owing to their construction with FCD. [28, 29] An
61
62 23
63
64
65
 independent report in literature backs our proposal by providing experimental evidence to FCDs

1 ability of killing breast cancer cells.[28]

2
3
4
Cell cycle arrest study affirmed the utility of PEI-FCD-DOX NPs when higher percentage
5
6 of cells stayed in G1-S phase along with a gradually increase in sub-G0/G1 phase population
7
8 pointing to enhancement in hypodiploids or apoptotic cell population. An apoptotic assay performed
9
10
11 using dual staining with Annexin V-FITC and propidium iodide dyes further suggested that
12
13 treatment with NPs caused greater apoptosis than necrosis. The raised apoptotic potential of PEI-
14
15
16 FCD-DOX NPs in comparison to free DOX points towards institution of an auxiliary supportive
17
18 mechanism by FCD, which traverses the age old inactive excipient cliché and synergizes with the
19
20
21 cytotoxic potential of DOX leading to probable reduction (if investigated further) in dose of DOX
22
23 and relatable toxicity.
24
25
Encouraged by the results obtained so far, we tried to clearly outline the instituting
26
27
28 principles behind the additional cytotoxicity of DOX loaded NPs. The most logical starting point
29
30 was investigating mitochondrial membrane potential perturbations after treating MDA-MB-231
31
32
33 cells with NPs, since DOX had already been reported to induce mitochondrial dysfunction
34
35 dependent cell death.[30] Our results enunciated that PEI-FCD-DOX NPs and PEI-FCD NPs
36
37
38 exhibited higher disruption of mitochondrial membrane potential compared to free DOX. The
39
40 enhanced mitochondrial membrane disruptive activity of NPs was laid down to the presence of
41
42
43
FCD, which induces mitochondrial dependent apoptosis. [31]
44
45 To assess immunotherapeutic potential of NPs during in vitro conditions, they were
46
47 incubated with co-culture of macrophages and cancer cells. NPs modulated the macrophage
48
49
50 differentiation and reduced the population of M2 phenotype of macrophages. Th-1 immune
51
52 response was enhanced upon NPs treatment indicated by improved secretion of IL-12 whereas Th-2
53
54
55 immune response was downregulated suggested by reduced secretion of IL-4. These results would
56
57 be attributed due to incorporation of FCD in the NPs. It has been reported that FCD activated the
58
59
60 macrophages during in vitro condition through scavenger receptors and induced Th1-1 immune
61
62 24
63
64
65
 response via releasing proinflammatory cytokines and nitric oxide[32, 33]. The differentiation of

1 macrophages towards M1 phenotypes would be facilitated directly through FCD or by secreted Th-1
2
3
4
immune cytokine i.e. IL-12 which is reported to activate macrophages through secretion of IFN-γ,
5
6 TNF-α and nitric oxide[34, 35].
7
8 In vivo studies were performed in 4T1 induced tumor bearing mice to confirm the previous
9
10
11 in vitro results. In vivo antiproliferative study predicted an improved in vivo efficacy of PEI-FCD-
12
13 DOX NPs compared to free DOX and as expected PEI-FCD NPs also had very slight anticancer
14
15
16 activity. The results could be attributed to synergistic activity of FCD although other assistive
17
18 mechanisms could also be operational. Small particle size of the NPs would favor selective
19
20
21 accumulation of the DOX in tumor microenvironment due to EPR effect.[36] The, in vivo anticancer
22
23 study thus professes that PEI-FCD-DOX NPs could be an appropriate drug delivery system of DOX
24
25
to treat cancers having high density microvessels such as breast tumor.
26
27
28 NPs exhibited an immunotherapeutic effect through enhanced secretion of IL-12 which
29
30 facilitates a Th-1 immune response to eradicate tumor growth and itself has been reported as a
31
32
33 potent anticancer agent in preclinical models.[37, 38] IL-12 is reported to activate natural killer
34
35 cells[39, 40] and natural killer cells facilitate a granzyme B, IFN-γ and death receptor mediated cell
36
37
38 death[41, 42]. Plasma IL-4 levels were decreased after treating animals with PEI-FCD-DOX NPs
39
40 and PEI-FCD NPs, proposing downregulation of Th-2 immune response. IL-4 is known to reduce
41
42
43
the production of proinflammatory cytokines (IL-12 and TNF-α) through monocytes and
44
45 macrophages leading to blockage of Th-1 immune response[43]. Upregulation of Th-1 immune
46
47 response and downregulation of Th-2 immune response by the NPs would lead to selective
48
49
50 differentiation of the TAMs towards M1 phenotype. Balancing of Th-1 and Th-2 immune response
51
52 would cause reversal of inflammatory cascades leading to tumor rejection based upon immune
53
54
55 system. The immunotherapeutic activity along with modulation of TAMs differentiation showed by
56
57 PEI-FCD NPs would be facilitated by the FCD through providing immunological responses. FCD is
58
59
60 known to activate the macrophages via carbohydrate receptors (fucoidan or scavenger receptors) to
61
62 25
63
64
65
 induce an immune response, which would in turns differentiate the macrophages towards M1

1 phenotype. FCD is reported to upregulate expression of CD40, CD80 and CD86 and enhanced
2
3
4
secretion of proinflammatory cytokines (IL-12 and TNF-α) during in vitro conditions in dendritic
5
6 cells. FCD defended animals challenged with cancer cells via eliciting Th-1 immune response
7
8 through modulating dendritic cells differentiation and cytotoxic T lymphocyte activation [44]. FCD
9
10
11 can even overcome the immunosuppressive effect of chemotherapeutics such as 5-fluorouracil.[10]
12
13 This immunomodulatory activity of the NPs loaded with chemotherapeutic agent would open a new
14
15
16 avenue against immuno coupled chemotherapy of breast cancer. Progression of breast tumor is
17
18 definitely effected by representative immune cells available in tumor microenvironment (TAMs)
19
20
21 and based on pharmacokinetic evidence presented below, it is anticipated NPs would accumulate in
22
23 tumor due to EPR effect. However, detailed studies are needed to explore mechanism behind the
24
25
modulating of TAMs differentiation by FCD. Figure S9, Supporting information, explains the
26
27
28 hypothetical molecular pathway behind the chemoimmunotherapy furnished by the developed NPs.
29
30 Serum DOX profile revealed that Cmax of DOX contained inside NPs was lower than that for
31
32
33 free DOX. However, the serum profile of DOX obtained post NPs administration was marked by
34
35 two crests. The initially latent maxima (occurs at around 1 h) in plasma can be ascribed to the initial
36
37
38 burst release offered by NPs as observed in dissolution studies. The latency and the suppressed
39
40 nature of the primary maxima is a direct consequence of the control executed by NPs in releasing
41
42
43
DOX. Although contradictory to the burst release phenomenon explained in the sentence preceding
44
45 last, it is mandatory to realize that NPs will always preclude unnecessary drug expulsion, and
46
47 therefore delay attainment of maximum plasma concentration, at least to some extent. The rapid fall
48
49
50 in concentration of DOX, after the initial maxima is a resultant of three distributive forces. Firstly,
51
52 as observed in profile of plane DOX, the drug released by NPs in plasma would readily equilibrate
53
54
55 in the entire body; secondarily routine metabolism and excretion will also reduce blood levels; and
56
57 thirdly owing to their size many DOX bearing NPs will either be phagocytosed or would penetrate
58
59
60 the perforated capillaries of major organs such as liver or the tumor itself. The extent to which these
61
62 26
63
64
65
 mutually inclusive factors contribute in the above stated dispositional processing requires further

1 investigations. Once inside the interstitium or intracellular spaces, the NPs are expected to slowly
2
3
4
release DOX, which under the influence of concentration gradient would effuse back into the
5
6 systemic circulation and generate secondary maxima as obtained at approximately 6 h and continue
7
8 to persist in serum for up to 24 h. The higher t1/2, and lower clearance along with the lower Ke of
9
10
11 DOX when it is packaged as NPs bears testimony to the above description. Overall, NPs exhibited
12
13 almost 3 folds increase in AUC. Biodistribution studies showed that higher amount of DOX was
14
15
16 present in liver, spleen and tumor when administered as NPs. It is safe to assume that the EPR effect
17
18 exhibited by NPs was quite possibly responsible for the relatively higher levels of DOX in tumor,
19
20
21 and will surely result in improved antiproliferative activity (a statement backed by evidences
22
23 presented above) in comparison to drug. Also, as explained previously, these higher levels would be
24
25
responsible for secondary elevated phase of DOX. On the same note, the concentration of DOX
26
27
28 when delivered as NP in kidney and heart was lower than that of free DOX suggesting reduced
29
30 chance of cardiac or related toxicity. Further, results of acute toxicity study support our hypothesis
31
32
33 that NPs reach their target site intact slowly releasing DOX thereby avoiding possibility of toxicity.
34
35 4. Conclusion
36
37
38 DOX encapsulated NPs were successfully prepared through PEC. NPs produced sustained release
39
40 drug profile. FCD NPs could positively modify the intracellular activities of DOX including
41
42
43
cytotoxicity, cell cycle arrest and apoptosis. The enhanced efficacy of the NPs was due to
44
45 mitochondrial depolarization mediated caspase dependent apoptosis. Furthermore, FCD arbitrated
46
47 an IL-12 driven Th1 immune response to retard tumor growth. In conclusion, developed NPs could
48
49
50 furnish immuno coupled chemotherapy for effective treatment of breast cancer along with improved
51
52 safety profile of the drug.
53
54
55 4.0 Experimental Section
56
57 See supporting information
58
59
60
61
62 27
63
64
65
 Supporting Information

1 Supporting Information is available from the Wiley Online Library or from the author.
2
3
4
Acknowledgement
5
6 This work was financially supported by grant from Council of Scientific and Industrial Research,
7
8 New Delhi, India under network project CSC0302. We are highly thankful to sophisticated
9
10
11 analytical facility, CDRI, Lucknow for facilitating flowcytometry facility. V.K.P. acknowledges
12
13 Indian Council of Medical Research, New Delhi, India for senior research fellowship. Y.S, J.G.P
14
15
16 and P.S acknowledge Council of Scientific and Industrial Research, New Delhi, India for senior
17
18 research fellowship. K.S acknowledges Department of Science and Technology, New Delhi, India
19
20
21 for senior research fellowship.
22
23 Conflict of interest
24
25
Authors declare no conflict of interest.
26
27
28 References
29 1. Porter, D. L.; Levine, B. L.; Kalos, M.; Bagg, A.; June, C. H., New England Journal of Medicine 2011,
30 365 (8), 725-733. DOI doi:10.1056/NEJMoa1103849.
31 2. Hee Lee, J.; Park, M.-S.; Hwang, J.-E.; Cho, S.-H.; Bae, W.-K.; Shim, H.-J.; Kim, D.-E.; Chung, I.-J., Cell
32
33 Mol Immunol 2013, 10 (3), 275-282. DOI 10.1038/cmi.2012.74.
34 3. Thomas, A.; Jakopovic, M., Expert Opinion on Biological Therapy 2014, 14 (8), 1061-1064. DOI
35 doi:10.1517/14712598.2014.925874.
36 4. Sharma, M. R.; Schilsky, R. L., Nat Rev Clin Oncol 2012, 9 (4), 208-214.
37 5. Zhang, T.; Herlyn, D., Cancer Immunol Immunother 2009, 58 (4), 475-492. DOI 10.1007/s00262-008-
38
39
0598-y.
40 6. Baars, A.; Claessen, A. M. E.; Wagstaff, J.; Giaccone, G.; Scheper, R. J.; Meijer, S.; Schakel, M. J. A. G.;
41 Gall, H. E.; Meijer, C. J. L. M.; Vermorken, J. B.; Pinedo, H. M.; van den Eertwegh, A. J. M., Br J Cancer 2002,
42 86 (8), 1230-1234.
43 7. Liu, Z.; Jiao, Y.; Wang, Y.; Zhou, C.; Zhang, Z., Advanced Drug Delivery Reviews 2008, 60 (15), 1650-
44
1662. DOI http://dx.doi.org/10.1016/j.addr.2008.09.001.
45
46 8. Pawar, V. K.; Panchal, S. B.; Singh, Y.; Meher, J. G.; Sharma, K.; Singh, P.; Bora, H. K.; Singh, A.; Datta,
47 D.; Chourasia, M. K., Journal of Controlled Release 2014, 196 (0), 295-306. DOI
48 http://dx.doi.org/10.1016/j.jconrel.2014.10.010.
49 9. Fitton, J. H., Marine Drugs 2011, 9 (10), 1731-1760. DOI 10.3390/md9101731.
50 10. Miyazaki, Y.; Nakamizo, M.; Shibasaki, T.; Kirino, T.; Kawahara, K.; Otsuka, K.; Tachibana, H.;
51
52 Yamada, K.; Tachikawa, D., The Journal of Immunology 2014, 192 (1 Supplement), 203.21.
53 11. Senthilkumar, K.; Kim, S.-K., Chapter Eleven - Anticancer Effects of Fucoidan. In Advances in Food
54 and Nutrition Research, Se-Kwon, K., Ed. Academic Press: 2014; Vol. Volume 72, pp 195-213.
55 12. Golfieri, R.; Giampalma, E.; Renzulli, M.; Cioni, R.; Bargellini, I.; Bartolozzi, C.; Breatta, A. D.; Gandini,
56 G.; Nani, R.; Gasparini, D.; Cucchetti, A.; Bolondi, L.; Trevisani, F., Br J Cancer 2014, 111 (2), 255-264. DOI
57
10.1038/bjc.2014.199.
58
59 13. Sledge, G. W.; Neuberg, D.; Bernardo, P.; Ingle, J. N.; Martino, S.; Rowinsky, E. K.; Wood, W. C.,
60 Journal of Clinical Oncology 2003, 21 (4), 588-592. DOI 10.1200/jco.2003.08.013.
61
62 28
63
64
65
 14. Keizer, H. G.; Pinedo, H. M.; Schuurhuis, G. J.; Joenje, H., Pharmacology & Therapeutics 1990, 47 (2),
219-231. DOI http://dx.doi.org/10.1016/0163-7258(90)90088-J.
1 15. Park, J.; Fong, P. M.; Lu, J.; Russell, K. S.; Booth, C. J.; Saltzman, W. M.; Fahmy, T. M., Nanomedicine:
2 Nanotechnology, Biology and Medicine 2009, 5 (4), 410-418. DOI
3 http://dx.doi.org/10.1016/j.nano.2009.02.002.
4
5
16. Yuan, A.; Wu, J.; Song, C.; Tang, X.; Qiao, Q.; Zhao, L.; Gong, G.; Hu, Y., Journal of Pharmaceutical
6 Sciences 2013, 102 (5), 1626-1635. DOI 10.1002/jps.23455.
7 17. Wang, A. Z.; Langer, R.; Farokhzad, O. C., Annual Review of Medicine 2012, 63 (1), 185-198. DOI
8 doi:10.1146/annurev-med-040210-162544.
9 18. Poon, W.; Zhang , X.; Nadeau, J., 2014, 19 (3-4), 223-245. DOI 10.1615/CritRevOncog.2014011563.
10
19. Davis, M. E.; Chen, Z.; Shin, D. M., Nat Rev Drug Discov 2008, 7 (9), 771-782.
11
12 20. Schroeder, A.; Heller, D. A.; Winslow, M. M.; Dahlman, J. E.; Pratt, G. W.; Langer, R.; Jacks, T.;
13 Anderson, D. G., Nat Rev Cancer 2012, 12 (1), 39-50.
14 21. Pawar, V. K.; Singh, Y.; Meher, J. G.; Gupta, S.; Chourasia, M. K., Journal of Controlled Release 2014,
15 183 (0), 51-66. DOI http://dx.doi.org/10.1016/j.jconrel.2014.03.030.
16 22. Arvizo, R. R.; Miranda, O. R.; Moyano, D. F.; Walden, C. A.; Giri, K.; Bhattacharya, R.; Robertson, J.
17
18 D.; Rotello, V. M.; Reid, J. M.; Mukherjee, P., PloS one 2011, 6 (9), e24374. DOI
19 10.1371/journal.pone.0024374.
20 23. Bronte, V.; Murray, P. J., Nat Med 2015, 21 (2), 117-119. DOI 10.1038/nm.3794.
21 24. Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R., Nat Nano 2007, 2 (12), 751-
22 760.
23
25. Ge, Z.; Liu, S., Chemical Society Reviews 2013, 42 (17), 7289-7325. DOI 10.1039/C3CS60048C.
24
25 26. Kozlovskaya, V.; Alexander, J. F.; Wang, Y.; Kuncewicz, T.; Liu, X.; Godin, B.; Kharlampieva, E., ACS
26 Nano 2014, 8 (6), 5725-5737. DOI 10.1021/nn500512x.
27 27. Poon, Z.; Chang, D.; Zhao, X.; Hammond, P. T., ACS Nano 2011, 5 (6), 4284-4292. DOI
28 10.1021/nn200876f.
29 28. Zhang, Z.; Teruya, K.; Yoshida, T.; Eto, H.; Shirahata, S., Marine Drugs 2013, 11 (1), 81-98.
30
31 29. Foley, S. A.; Szegezdi, E.; Mulloy, B.; Samali, A.; Tuohy, M. G., Journal of Natural Products 2011, 74
32 (9), 1851-1861. DOI 10.1021/np200124m.
33 30. Kuznetsov, A. V.; Margreiter, R.; Amberger, A.; Saks, V.; Grimm, M., Biochimica et Biophysica Acta
34 (BBA) - Molecular Cell Research 2011, 1813 (6), 1144-1152. DOI
35 http://dx.doi.org/10.1016/j.bbamcr.2011.03.002.
36
37
31. Zhang, Z.; Teruya, K.; Eto, H.; Shirahata, S., PloS one 2011, 6 (11), e27441. DOI
38 10.1371/journal.pone.0027441.
39 32. Hsu, H.-Y.; Chiu, S.-L.; Wen, M.-H.; Chen, K.-Y.; Hua, K.-F., Journal of Biological Chemistry 2001, 276
40 (31), 28719-28730. DOI 10.1074/jbc.M011117200.
41 33. Teruya, T.; Tatemoto, H.; Konishi, T.; Tako, M., Glycoconjugate journal 2009, 26 (8), 1019-28. DOI
42
10.1007/s10719-008-9221-x.
43
44 34. Xing, Z.; Zganiacz, A.; Santosuosso, M., Journal of leukocyte biology 2000, 68 (6), 897-902.
45 35. Grohmann, U.; Belladonna, M. L.; Vacca, C.; Bianchi, R.; Fallarino, F.; Orabona, C.; Fioretti, M. C.;
46 Puccetti, P., The Journal of Immunology 2001, 167 (1), 221-227. DOI 10.4049/jimmunol.167.1.221.
47 36. Meng, H.; Xue, M.; Xia, T.; Ji, Z.; Tarn, D. Y.; Zink, J. I.; Nel, A. E., ACS Nano 2011, 5 (5), 4131-4144.
48 DOI 10.1021/nn200809t.
49
50 37. Lasek, W.; Zagożdżon, R.; Jakobisiak, M., Cancer Immunol Immunother 2014, 63 (5), 419-435. DOI
51 10.1007/s00262-014-1523-1.
52 38. Del Vecchio, M.; Bajetta, E.; Canova, S.; Lotze, M. T.; Wesa, A.; Parmiani, G.; Anichini, A., Clinical
53 Cancer Research 2007, 13 (16), 4677-4685. DOI 10.1158/1078-0432.ccr-07-0776.
54 39. Lauwerys, B. R.; Renauld, J. C.; Houssiau, F. A., Cytokine 1999, 11 (11), 822-30. DOI
55
10.1006/cyto.1999.0501.
56
57 40. D'Andrea, A.; Rengaraju, M.; Valiante, N. M.; Chehimi, J.; Kubin, M.; Aste, M.; Chan, S. H.;
58 Kobayashi, M.; Young, D.; Nickbarg, E.; et al., J Exp Med 1992, 176 (5), 1387-98.
59
60
61
62 29
63
64
65
 41. Ida, H.; Nakashima, T.; Kedersha, N. L.; Yamasaki, S.; Huang, M.; Izumi, Y.; Miyashita, T.; Origuchi, T.;
Kawakami, A.; Migita, K.; Bird, P. I.; Anderson, P.; Eguchi, K., European journal of immunology 2003, 33 (12),
1 3284-92. DOI 10.1002/eji.200324376.
2 42. Smyth, M. J.; Hayakawa, Y.; Takeda, K.; Yagita, H., Nat Rev Cancer 2002, 2 (11), 850-861.
3 43. Levings, M. K.; Schrader, J. W., The Journal of Immunology 1999, 162 (9), 5224-5229.
4
5
44. Jin, J. O.; Zhang, W.; Du, J. Y.; Wong, K. W.; Oda, T.; Yu, Q., PloS one 2014, 9 (6), e99396. DOI
6 10.1371/journal.pone.0099396.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 30
63
64
65
Supporting Information
Click here to download Supporting Information: Supporting information.docx