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Hypoxia responsive, tumor penetrating lipid nanoparticles for delivery of chemotherapeutics to pancreatic cancer cell spheroids Prajakta Kulkarni, Manas K Haldar, Preeya Katti, Courtney Dawes, Seungyong You, Yongki Choi, and Sanku Mallik Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00241 • Publication Date (Web): 08 Jul 2016 Downloaded from http://pubs.acs.org on July 11, 2016

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

TOC Graphic:

ACS Paragon Plus Environment


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1

Hypoxia responsive, tumor penetrating lipid nanoparticles for

2

delivery of chemotherapeutics to pancreatic cancer cell spheroids

3

Prajakta Kulkarni1, Manas K. Haldar1, Preeya Katti2, Courtney Dawes3, Seungyong You4,

4

Yongki Choi4, Sanku Mallik1

5

1

6

58102

7

2

Davies High school, Fargo, North Dakota 58104

8

3

Valley City High school, Valley City, North Dakota 58072

9

4

Department of Physics and Mathematics, North Dakota State University, Fargo, North Dakota

10

Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota

58102

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12 13 14 15 16 17 18 19 20 21


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Abstract:

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The solid tumors are often poorly irrigated due to the structurally compromised microcirculation.

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Uncontrolled multiplication of cancer cells, insufficient blood flow, and the lack of enough oxygen

25

and nutrients lead to the development of hypoxic regions in the tumor tissues. As the partial

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pressure of oxygen drops below the necessary level (10 psi), the cancer cells modulate their genetic

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makeup to survive. Hypoxia triggers tumor progression by enhancing angiogenesis, cancer stem

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cell production, remodeling of the extracellular matrix, and epigenetic changes in the cancer cells.

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However, the hypoxic regions are usually located deep in the tumors and are usually inaccessible

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to the intravenously injected drug carrier or the drug. Considering the designs of the reported

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nanoparticles, it is likely that the drug is delivered to the peripheral tumor tissues, close to the

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blood vessels. In this study, we prepared lipid nanoparticles (LNs) comprising the synthesized

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hypoxia-responsive lipid and a peptide-lipid conjugate. We observed that the resultant LNs

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penetrated to the hypoxic regions of the tumors. Under low oxygen partial pressure, the hypoxia-

35

responsive lipid undergoes reduction, destabilize the lipid membrane, and release the encapsulated

36

drugs from the nanoparticles. We demonstrated the results employing spheroidal cultures of the

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pancreatic cancer cells BxPC-3. We observed that the peptide-decorated, drug encapsulated LNs

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reduced the viability of pancreatic cancer cells of the spheroids to 35% under hypoxic conditions.

39

Keywords:

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Hypoxia responsive LNs, stimuli-responsive nanoparticles, spheroidal cultures, iRGD peptide

41

Introduction:

42

Stimuli-responsive nanoparticles show tremendous potential to deliver anticancer

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chemotherapeutics to the targeted tumor site with enhanced efficacy and reduced side effects.1



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Liposomes are the most popular lipid-based bilayer vesicles approved by the US Food and Drug

45

Administration (FDA) for the treatment of various cancers.2 Compared to the conventional

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chemotherapy, the anticancer drug encapsulated, polyethylene glycol-coated (PEGylated)

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liposomes show reduced side effects.3 However, the slow release of the encapsulated drugs from

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the clinically-approved liposomal formulations have stimulated intense research on tumor

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microenvironment responsive, PEGylated drug carriers.

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After intravenous administration, the PEGylated nanoparticles accumulate in the tumor

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tissues due to the leaky vasculature and poor lymphatic drainage (the Enhanced Permeation and

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Retention or EPR effect).4 The abnormal tumor microenvironment (such as, reduced pH, elevated

53

enzyme and reducing agent levels, high cell surface receptor density) acts as triggers for stimuli-

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responsive nanoparticles to release the encapsulated drugs.5 In-vivo studies demonstrate better

55

control over tumor growth by the drug-encapsulated, stimuli-responsive nanoparticles compared

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to the conventional treatment.6 Considering the designs of the reported nanoparticles, it is likely

57

that the drug is delivered to the peripheral tumor tissues, close to the blood vessels.

58

The solid tumors are often poorly irrigated due to the structurally compromised

59

microcirculation.7 Uncontrolled multiplication of cancer cells, insufficient blood flow, and the lack

60

of enough oxygen and nutrients lead to the development of hypoxic regions in the tumor tissues.

61

As the partial pressure of oxygen drops below the necessary level (10 psi), the cancer cells

62

modulate their genetic makeup to survive.8 Hypoxia triggers tumor progression by enhancing

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angiogenesis, cancer stem cell production, remodeling of the extracellular matrix, and epigenetic

64

changes in the cancer cells.9 However, the hypoxic regions are usually located deep in the tumors

65

and are usually inaccessible to the intravenously injected drug carrier or the drug.10 The cyclic


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iRGD peptide is reported to interact with the overexpressed integrin and neuropilin receptors11 –

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increasing the penetration of nanoparticles in tumor tissues.12

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In this study, we prepared LNs comprising a synthesized hypoxia-responsive, PEGylated

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lipid and an iRGD peptide conjugated lipid. We hypothesized that the resultant LNs would

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penetrate to the hypoxic regions of the tumors. Under low oxygen partial pressure, the hypoxia

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responsive lipid will undergo reduction, destabilize the lipid membrane, and release the

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encapsulated drugs from the LNs. We validated our hypothesis employing spheroidal cultures of

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the pancreatic cancer cells BxPC-3. The BxPC-3 cells form coherent spheroids with hypoxic cores

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– making them ideal for in-vitro studies.13 We observed that the iRGD peptide-decorated, drug

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encapsulated LNs reduced the viability of pancreatic cancer cells of the spheroids to 35% under

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hypoxic conditions. To the best of our knowledge, this is the first report of the hypoxia-triggered

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release of liposomal anticancer drugs.

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Results and discussion:

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Hypoxia alters the tumor microenvironment through the overexpressed HIF-1 and contributes to

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the overall cancer progression and metastasis.14,15 The azobenzene group is reduced in the presence

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of elevated levels of reducing enzymes in the hypoxic tumor microenvironment (Figure 1).16 We

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synthesized a hypoxia-sensitive lipid by conjugating POPE to polyethylene glycol (PEG1900)

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through the azobenzene linker (Scheme 1). We incubated a solution of the purified lipid under

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simulated hypoxic conditions (100 M NADPH, rat liver microsomes, and nitrogen gas bubbling)

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for 2 hours. The rat liver microsomes contain cytochrome P450 enzymes.17 Reduction of

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azobenzene takes place under hypoxic conditions in the presence of reducing enzymes and a

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hydrogen donor source. Hence, for in-vitro studies we have used rat liver microsomes as a source



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of reducing enzymes and NADPH as a proton donor for the reduction of azobenzene. We observed

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that the fluorescence emission intensity from the azobenzene group was substantially reduced after

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the hypoxic treatment. We did not observe any reduction in the emission intensity of the lipid

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under normoxic conditions (100 M NADPH, rat liver microsomes, and nitrogen gas bubbling).

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Scheme 1. Synthesis of the hypoxia responsive POPE-azobenzene-PEG1900 lipid. The hypoxia-

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responsive linker is indicated in red.


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Figure 1. Mechanism of reduction of azobenzene under hypoxic reducing environment, where R

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represents POPE.16

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Tumor tissues develop hypoxia in the regions with inadequate blood flow due to the

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irregularly formed vasculature.15 These areas are usually less accessible to passively targeted drug

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carriers. The cyclic iRGD peptide has been previously used to enhance accumulation of the drugs

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or nanoparticles in the tumor tissues.12,

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preferentially accumulate and penetrate deep into the tumor tissue.19

18

The iRGD peptide functionalized nanoparticles

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We synthesized the hexynoic acid conjugated iRGD peptide by employing a microwave

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assisted, solid-phase peptide synthesizer. The alkyne of hexynoic acid allowed conjugation of the

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synthesized peptide to the surface of the LNs by the “Click” chemistry. To confirm the cyclization

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of the peptide, we recorded the CD spectrum in the absence and presence of 10 mM glutathione.

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Thiol-sulfhydryl exchange and the reduction of the disulfide bond in the peptide resulted in a shift

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of the negative peak in the CD spectra from 199 nm to 195 nm (Supporting Information, Figure



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S2). The peptide was then conjugated to the saturated lipid DSPE-PEG-N3 by using the copper

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catalyzed [2+3]-cycloaddition reaction (Scheme 2). The product was confirmed by CD

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spectroscopy (Supporting Information, Figure S3). This lipid was then incorporated into the LNs

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to enhance the tissue penetration depth of the vesicles.

113 114

Scheme 2. Synthesis of iRGD peptide conjugated DSPE lipid. The iRGD peptide is shown in

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blue.

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The stability of the nanoparticles in the absence of a stimulus is critical for drug delivery

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applications. We have previously observed that liposomes prepared from a proper combination of

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unsaturated and saturated lipids are structurally stable in the circulation, although the lipids phase-

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separate in the bilayer. However, in the presence of a stimulus, the vesicles become unstable and

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rapidly release the contents.6 Based on the precedent, we prepared the LNs incorporating DSPC,

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DSPE-PEG-iRGD, and the hypoxia-sensitive lipid containing the unsaturated POPE group. We


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optimized the relative ratio of these lipids such that the LNs are stable in normoxic circulatory

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conditions but disintegrate in the hypoxic environment to release the encapsulated contents. We

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encapsulated the self-quenching dye carboxyfluorescein in the LNs and monitored its release

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employing fluorescence spectroscopy (excitation: 485 nm, emission: 515 nm). The optimum

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stability and release characteristics were observed with the lipid composition DSPC: POPE-

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azobenzene-PEG1900: DSPE-PEG-iRGD in the molar percentage of 40:50:10. Under normoxic

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conditions, the release from these LNs was observed to be less than 5% in 2 hours (Figure 2, black

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squares). However, under hypoxic conditions, the LNs released 66% of the encapsulated dye

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within 2 hours (Figure 2, red triangles). As another control, we prepared carboxyfluorescein-

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encapsulated liposomes from the DSPC lipid and subjected them to the hypoxic conditions. We

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observed less than 20% of contents release in 2 hours (Figure S4, Supporting Information). We

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anticipate that the reduction of the azobenzene linker will separate the PEG and the POPE

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fragments of the hypoxia-sensitive lipid (Scheme 1). The resultant shift in the hydrophilic-

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hydrophobic balance of the lipids will likely destabilize the nanoparticles, leading to the release of

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the encapsulated contents.6



70 60 Percent release

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50 40 30 20 10 0 0

20

40

60

80

100

120

Time (min)

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Figure 2. Dye release profiles from the LNs in hypoxic (red triangles) and normoxic (black

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squares) environment. The lines connecting the observed data points are shown (n = 3).

140 141

The LNs were less than 250 nm in size with a polydispersity index (PDI) of less than 0.3.

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A typical size distribution pattern, as measured by dynamic light scattering, is shown in Figure 3.

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Table 1 shows the size analysis of the LNs as determined by dynamic light scattering.


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16 14 Percent intensity

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12 10 8 6 4 2 0 0

200

400

600

800

1000

Size (nm)

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Figure 3. Size distribution of LNs as determined by the dynamic light scattering. The mean

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diameter was (180 + 3) nm with a polydispersity index of 0.23 + 0.01.

147 148

Table 1. Size analysis by dynamic light scattering for the test and the control LNs. Lipid composition (molar percentage)

Encapsulated content

DSPC

DSPC (100)

Test LNs

POPE-azobenzene-PEG (50): DSPC (39): DSPE-PEG-iRGD (10): Lissamine rhodamine (1) POPE-AZB-PEG (50): DSPC (49): Lissamine rhodamine (1) POPE-AZB-PEG (50): DSPC (40): DSPE-PEG-iRGD (10):

LNs without iRGD

Test LNs

PDI ± std. dev.

gemcitabine

size (nm) ± std. dev. 178 ± 3.6

gemcitabine

180 ± 3.4

0.23 ± 0.01

gemcitabine

121 ± 1.5

0.12 ± 0.01

carboxyfluorescein

235 ± 2.3

0.20 ± 0.04

0.15 ± 0.02

149 150 151

We imaged the LNs by atomic force microscopy (AFM) to determine any structural

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changes in the hypoxic environment. In this endeavor, we prepared the LNs in 25 mM HEPES

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buffer (pH 7.4) and added NADPH and rat liver microsomes. The control samples were exposed



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to normal atmospheric oxygen levels while nitrogen gas was bubbled through the test reaction

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mixture for 2 hours. We observed that the LNs under normoxic conditions retained the spherical

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structures (Figure 4, Panel A). However, hypoxic conditions distorted the shapes of the vesicles

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(Figure 4, Panel B), indicating structural changes in the lipid bilayer. We conducted the AFM

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studies by placing the LNs on a mica surface, drying, and subsequent imaging. We note that some

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inadvertent structural perturbations may be introduced due to the sample preparation procedure.

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However, multiple reports indicate that liposomes and other lipid nanoparticles retain their

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structure after drying on a mica surface.20,21

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A

B

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Figure 4. AFM images of LNs under normal oxygen levels (A) and after 2 hours of hypoxia

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treatment in the presence of NADPH and rat liver microsomes (B).


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After confirming the hypoxia responsive characteristics of the LNs, we encapsulated the

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anticancer drug gemcitabine by the active loading method (as a model hydrophilic drug).6

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Gemcitabine is the first line of treatment for pancreatic cancer. Since gemcitabine in a solution is

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colorless, we incorporated 1% of the fluorescent lipid 1,2-dipalmitoyl-sn-glycero-3-

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phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (lissamine rhodamine lipid) in the LNs

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(molar ratio of DSPC: POPE-azobenzene-PEG1900: DSPE-PEG-iRGD: lissamine rhodamine lipid

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39:50:10:1) for visualization. To encapsulate gemcitabine, a pH gradient was developed across

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the lipid bilayer with inside pH of 4 and outside pH of 7.4. After the active loading, we used gel

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filtration to remove any unencapsulated drug. The entrapment efficiency for these LNs was found

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to be about 40% with a drug loading capacity of 8% (by absorption spectroscopy).

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The effectiveness of the drug encapsulated LNs was tested using monolayer and three-

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dimensional spheroid cultures of the pancreatic cancer cells BxPC-3. We observed that the

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viability of cells in monolayer cultures decreased in the presence of LNs encapsulating

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gemcitabine under both normoxic and hypoxic conditions. However, the response of three-

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dimensional tumor tissues cannot be predicted from monolayer cells cultures.22 Hence, we

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cultured the BxPC-3 cells as spheroids in agarose scaffolds (Figure 5).



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Figure 5. Optical microscopic images of the cultured spheroids of BxPC-3 cells in agarose molds

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at (A) 4X magnification (scale bar: 200 µm), and (B) 10X magnification (scale bar: 50 µm).

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We divided the BxPC-3 cell spheroids into two groups and cultured them in normoxic and

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hypoxic (1% oxygen) conditions for 72 hours. Both the groups received treatments with

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gemcitabine

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encapsulating LNs without the iRGD peptide on the surface, free gemcitabine, and gemcitabine

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encapsulating LNs devoid of hypoxia responsive lipid (prepared with DSPC). We measured the

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cell viability by the Alamar Blue assay.6 To make a valid comparison between the groups, we

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ensured the gemcitabine concentration was 20 µM for all the treatments. Results indicated that

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the cell viability in monolayer cultures (Figure 6A) was unaffected after treatment with drug

195

encapsulated LNs under normoxic and hypoxic conditions. However, we observed significant

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decrease in cell viability for the cells treated with iRGD conjugated LNs under hypoxia compared

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to the normoxic conditions (p ≤ 0.05). Cell viability in spheroids treated with DSPC LNs, free

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gemcitabine, and LNs devoid of iRGD peptide showed decrease in cell viability under normoxic

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as well as hypoxic conditions (Figure 6B). The reduction in cell viability under normoxic spheroids

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may be due to reduction of azobenzene and release of gemcitabine in hypoxic core of the BxPC-3

encapsulating

iRGD

functionalized

hypoxia-sensitive


LNs,

gemcitabine

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201

cell spheroids. The treatment with iRGD conjugated, hypoxia-responsive LNs decreased the cell

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viability in hypoxic spheroidal culture to 25% (Figure 6B) showing significant decrease in cell

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viability compared to the spheroidal cultures treated under normoxic conditions. We speculate that

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the enhanced cytotoxicity is due to the penetration ability of iRGD conjugated LNs. As the LNs

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penetrate deep inside the core of the spheroids, they are likely to encounter a hypoxic

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microenvironment and release the encapsulated drugs. The reduced effectiveness of the other

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liposomal formulations and the free gemcitabine is probably due to less penetration in the

208

spheroids.

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100 Percent Cell Viability

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A

80 60 40 20 0 DSPC Gemcitabine liposomes

Liposomes Liposomes (with iRGD) (no iRGD)

210



100

Percent cell viability

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B

80 60 40 20 0 DSPC Gemcitabine Liposomes Liposomes liposomes (no iRGD) (with iRGD)

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Figure 6. Viability of BxPC-3 cells cultured as monolayers (A) and three-dimensional spheroids

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(B) after treatment with DSPC LNs encapsulating gemcitabine (20 µM), free gemcitabine (20 µM),

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gemcitabine encapsulated hypoxia-responsive LNs devoid of surface iRGD peptide, and the

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hypoxia-sensitive LNs with the iRGD under normoxic (red bars) and hypoxic conditions (green

216

bars).

217 218

The cell viability studies in the spheroidal cultures indicated that the hypoxia-responsive LNs were

219

able to release the encapsulated drug in the hypoxic BxPC-3 cells. To observe the penetration

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depth facilitated by iRGD peptide on the surface, we cultured the BxPC-3 cells in a paper stack as

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a tumor tissue23 using our 3D printed apparatus (Materials and Methods). We delivered 20 L of

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carboxyfluorescein encapsulated, hypoxia-sensitive LNs through the hollow tube of the paper

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stack insert, and incubated for 2 hours under normoxic and hypoxic conditions. Subsequently, the

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apparatus was disassembled, the filter papers were separated, and imaged under a fluorescence

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microscope. We observed very low fluorescence under normoxic conditions after 2 hours, even


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from the top filter paper (Figure 7A, Normoxic Panel). However, under hypoxic conditions,

227

considerably higher fluorescence intensity was observed (Figure 7B, Hypoxic Panel) from the top

228

filter paper, indicating the release of the LN encapsulated carboxyfluorescein dye. We also noted

229

that the LNs conjugated to the iRGD peptide penetrated deeper into the cultured tumor tissues.

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LNs devoid of the iRGD peptide were unable to reach the 8th layer of the stack under hypoxic

231

conditions (Figure 7A, Hypoxic Panel). However, LNs functionalized with the iRGD peptide

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penetrated deeper into the stack, reaching the 12th layer of the culture from the top (approximately

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1.8 mm in depth, Figure 7B, Hypoxic Panel).

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Figure 7. Fluorescence microscopic images of layers of cells indicating carboxyfluorescein release

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from the LNs under normoxic and hypoxic conditions without (A) and with (B) the iRGD peptide

238

functionalization (scale bar: 100 µm).

239

In conclusion, we have successfully synthesized a hypoxia-sensitive lipid and prepared

240

iRGD peptide functionalized, hypoxia-responsive LNs encapsulating the anticancer drug

241

gemcitabine in the aqueous core. The LNs released 65% of the encapsulated contents under

242

hypoxic conditions in 2 hours. The pancreatic cancer cells BxPC-3 treated with the LNs showed

243

decreased cell viability compared to the free drug and DSPC LNs. The iRGD peptide on the surface

244

allowed the LNs to penetrate deeper and deliver the anticancer drug to the hypoxic cores, resulting

245

in enhanced cytotoxicity for the cultured pancreatic cancer cell spheroids.

246


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Materials and Methods:

248

Materials:

249

The amino acids and the resins for peptide synthesis were purchased from Peptides International.

250

The lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-

251

phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (rhodamine lipid), and

252

palmitoyl oleoyl phosphatidylethanolamine (POPE) were purchased from Avanti Polar Lipids.

253

The 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG-N3 (molecular weight 5,000)

254

was purchased from Nanocs. The pancreatic cancer cell line BxPC-3 was purchased from ATCC.

255

The cell culture media, fetal bovine serum, and antibiotics were purchased from Lonza. All other

256

chemicals used were laboratory grade.

257

Synthesis and characterization of hypoxia responsive lipid PEG–azobenzene–POPE:

258

Following a reported procedure,24 the amine terminated mPEG2000 was conjugated with the

259

azobenzene-4,4' dicarboxylic acid. NMR spectrum confirmed formation of the product.24 1H NMR

260

(400 MHz, CHLOROFORM-d) δ ppm 0.00 - 0.01 (m) 1.08 - 1.36 (m) 3.37 - 3.38 (m) 3.53 - 3.63

261

(m) 3.77 - 3.91 (m) 5.25 - 5.38 (m) 7.27 (s) 7.93 - 8.07 (m). The resultant Polymer attached

262

diphenyl azacarboxylate (100 mg, 0.046 mmol) was reacted with POPE (35 mg, 0.048 mmol)

263

employing HBTU (19 mg, 0.048 mmol), HOBt (7 mg, 0.050 mmol), and diisopropylethyl amine

264

(18 mg, 0.138 mmol) in DMF (20 mL) under inert conditions overnight. After removal of the

265

solvent under reduced pressure, the semisolid was dissolved in dichloromethane, washed with 10%

266

citric acid solution, and 0.5 N NaOH solution. The clear organic phase was dried over sodium

267

sulfate and evaporated under reduced pressure. The resulting semisolid was subjected to

268

chromatographic purification (Rf = 0.7 in 10% methanol in dichloromethane) affording 105 mg



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(79%) of the final product. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.80 - 0.92 (m) 1.16 -

270

1.40 (m) 1.52 - 1.64 (m) 1.97 - 2.22 (m) 2.24 - 2.32 (m) 2.91 - 3.08 (m) 3.35 - 3.50 (m) 3.54 - 3.75

271

(m) 3.90 - 4.07 (m) 4.09 - 4.22 (m) 5.19 - 5.38 (m) 7.27 (s) 7.91 - 8.03 (m). 13C NMR (100 MHz,

272

CHLOROFORM-d) δ ppm 14.12, 22.69, 27.22, 29.32, 29.53, 29.71, 45.60, 59.04, 70.57, 71.95,

273

76.71, 77.03, 77.35, 119.07, 128.51, 133.30, 153.08, 167.2, 173.79.

274

Synthesis and characterization of the iRGD peptide:

275

The peptide was synthesized using a microwave-assisted, solid-phase peptide synthesizer (Liberty

276

Blue with Discover microwave unit, CEM Corporation) employing the CLEAR Amide resin as

277

the solid support (0.2 g, 0.1 mmol/g). The sequence hexynoic acid-Cys(Acm)-Arg(Pbf)-Gly-

278

Asp(OBut)-Lys(Boc)-Gly-Pro-Asp(OBut)-Cys(Acm)-OH was synthesized without the final

279

deprotection. To cyclize the product, thallium trifluoroacetate (55 mg, 0.1 mmol) in DMF (5 mL)

280

was stirred with the resin conjugated protected peptide for 3 hours. The resin was washed with

281

DMF (3X) and dichloromethane (3X). Subsequently, the peptide was cleaved from the resin with

282

trifluoroacetic acid (19 mL), and distilled water (0.5 mL) for 2 hours. The resin was filtered

283

through a Whatman filter paper. To the filtrate, 15 mL cold diethyl ether was added, and the

284

obtained precipitate was dried in a vacuum desiccator. The obtained product was characterized by

285

MALDI-TOF mass spectrometry (Observed mass: 1042.36, Expected Mass: 1042.43, Supporting

286

Information, Figure S1) and circular dichroism spectroscopy. CD spectra were recorded using 1

287

mg/mL solution of the iRGD peptide in phosphate buffer (4 mM, pH = 7.5, Supporting

288

Information, Figure S2).

289

290


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291

Synthesis of iRGD peptide-lipid conjugate:

292

The lipid DSPE-PEG-N3 was reacted with the hexynoic acid conjugated iRGD peptide using the

293

“Click” reaction. To conjugate the alkyne of the peptide to the azide group of the lipid, the

294

compounds were reacted in 1:2 molar ratio. The hexynoic acid conjugated peptide (40 mg) and

295

DSPE-PEG-N3 (50 mg) were dissolved in 3 mL water. The copper complex was prepared by

296

mixing copper (II) sulfate with N, N, N’, N’, N’’-pentamethyl diethylenetriamine (PMDETA) for

297

2 hours at room temperature. The ascorbic acid solution was prepared in water. The copper

298

complex (53 mM CuSO4 solution in water and 2 mmol PMDETA stirred for 2 hours) and sodium

299

ascorbate (1.4 µmol) were added to the reaction mixture and stirred for 24 hours at room

300

temperature. The sample was then transferred to a dialysis cassette with a molecular weight cut-

301

off of 3,000. The reaction mixture was dialyzed against water for 72 hours to remove the catalyst

302

and unreacted iRGD peptide. The product was then analyzed by CD spectroscopy (Supporting

303

Information, Figure S3).

304

Preparation of LNs for release studies:

305

LNs were prepared by the thin film hydration method. DSPC, hypoxia-sensitive POPE-

306

azobenzene-PEG, and iRGD conjugated DSPE lipid were dissolved in chloroform in a round

307

bottom flask (molar ratio 50:40:10 respectively). Chloroform was then evaporated using a rotary

308

evaporator to form a thin film at the bottom of the flask. The thin film was dried overnight in a

309

vacuum desiccator. The dry film was hydrated with a carboxyfluorescein (100 mM) solution

310

(prepared in 25 mM HEPES buffer, pH 7.4) for 1 hour at 60 °C. The multi-lamellar LNs formed

311

were then subjected to ultrasonication for 1 hour at level 9 on a bath sonicator (Aquasonic, model

312

250D). The formed LNs were then extruded 11 times through 0.8 µm Whatman filter paper using



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Page 22 of 33

313

an extruder (Avanti Polar Lipids). To reduce the size, the LNs were again extruded 11 times

314

through 0.2 µm Whatman filter. Excess of carboxyfluorescein (unencapsulated) was removed by

315

a Sephadex G100 size exclusion column. The eluted LNs were used for release studies.

316

Size analysis:

317

The hydrodynamic diameter of the LNs was measured by dynamic light scattering, using a

318

Malvern Zetasizer. All measurements were performed employing 1 mL sample with 6 runs and 6

319

repeats.

320

Release studies:

321

LNs (20 µL of 1 mg/mL total lipid concentration) encapsulating carboxyfluorescein were taken in

322

25 mM HEPES buffer of pH 7.5 (180 µL). Rat liver microsomes were isolated and (10 µL of 790

323

µg/mL) were added to this solution along with NADPH (100 µM) as a cofactor.25 The LNs were

324

subjected to normoxic and hypoxic conditions to measure the release of the encapsulated dye.

325

Normoxic conditions were maintained by bubbling air through the buffer containing the LNs.

326

Hypoxic conditions were created by bubbling nitrogen through the reaction mixture. Emission

327

intensity of the mixture was measured at 515 nm as a function of time (excitation: 485 nm).

328

𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑟𝑒𝑙𝑒𝑎𝑠𝑒

329

=

(𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑎𝑓𝑡𝑒𝑟 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 − 𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑏𝑒𝑓𝑜𝑟𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒) × 100 (𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑎𝑓𝑡𝑒𝑟 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑤𝑖𝑡ℎ 𝑡𝑟𝑖𝑡𝑜𝑛 − 𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑏𝑒𝑓𝑜𝑟𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒)

330

Atomic force microscopic imaging:

331

An atomic force microscope (AFM) was used to image the LNs on a mica surface. The AFM

332

measurements were carried out in non-contact mode at a scanning rate of 0.7 Hz and a resonance

333

frequency of 145 kHz using an NT-MDT NTEGRA (NT-MDT America, Tempe, AZ). The


Page 23 of 33

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334

cantilever was made of silicon nitride and was 100 m long. The scanning areas were 5 x 5 or 20

335

x 20 m at the resolution of 512 or 1024 points per line, respectively. The LN solution was dropped

336

on top of a freshly cleaved mica surface and kept for 10 minutes at room temperature. The

337

remaining solution was rinsed with water and dried extensively with an air blow gun. The mica

338

substrate was glued on top of a sapphire substrate (sample holder) using Scotch double sided tape,

339

and cleaved with Scotch tape to obtain a debris-free and flat surface. The images were flattened

340

by a first order line correction and a first order plane subtraction to compensate for the sample tilt.

341 342

Preparation of gemcitabine encapsulated LNs:

343

Gemcitabine was encapsulated in LNs by the active loading method.6 The lipid DSPC, hypoxia-

344

sensitive POPE-azobenzene-PEG, iRGD conjugated DSPE, and POPE-lissamine rhodamine were

345

dissolved in chloroform in a round bottom flask (molar ratio of 49:40:10:1 respectively). The lipid

346

thin film was prepared as described before. The thin film was hydrated with 25 mM citrate buffer

347

(pH 4) for 1 hour at 60 °C. Subsequently, the formed LNs were sonicated in a bath sonicator

348

(Aquasonic 250D, Power level 9) for 1 hour. The LNs were then extruded through a 0.2 µm

349

Whatman Nucleopore membranes for 11 times and were passed through Sephadex G100 column

350

to build the pH gradient across the membrane. Gemcitabine was encapsulated in these LNs by

351

incubating the drug (lipid: drug ratio 9:1) with the LNs for 3 hours at 60 °C. The absorbance

352

spectrum of the LNs was recorded. From a standard curve, the amount of gemcitabine was

353

determined. Percent entrapment and encapsulation efficiency for gemcitabine were calculated

354

using the following formula:



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 49 50 51 52 53 54 55 56 57 58 59 60

355 356

357

Page 24 of 33

𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑒𝑥𝑡𝑟𝑎𝑝𝑚𝑒𝑛𝑡 =

gemcitabine before gel filtration (mg) − gemcitabine after gel filtration (mg) × 100 gemcitabine before gel filtration (mg)

𝐸𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 =

Gemcitabine encapsulated in LNs (mg/mL) × 100 Weight of of the lipids (mg/mL)

358

Cellular studies:

359

The cellular studies were carried out with the pancreatic cancer cell line BxPC-3 in RPMI media

360

(supplemented with 10% FBS and 1% antibiotics). The cells were grown as monolayer and as

361

three-dimensional spheroids. Spheroids grown on 96 well agarose microwell plate were used for

362

cell viability assay. To study the penetration ability of the LNs, BxPC-3 cells were grown on stacks

363

of wet strengthened Whatman filter paper (number 114).

364 365

Cell viability studies in monolayer cultures:

366

In a 96 well clear bottom plate, the BxPC-3 cells (2000 per well) were seeded. The cells were

367

allowed to attach to the surface and grow for 24 hours at 37 °C in an incubator supplemented with

368

5% CO2. The cells received treatment with hypoxia responsive, iRGD peptide-decorated LNs

369

encapsulating gemcitabine (20 µM) for 3 days. LNs devoid of hypoxia-sensitive lipid and without

370

iRGD conjugated lipid were used as controls. Gemcitabine (20 µM) in aqueous solution was used

371

as a positive control. After 3 days of treatment, the conditioned media was removed from the plate

372

(to discard the fluorescent LNs) and was replaced with 200 µL of fresh medium supplemented

373

with 20 µL Alamar Blue (Invitrogen). The plates were incubated for 2 hours, and fluorescence was

374

measured (excitation wavelength 560 nm, emission wavelength 590 nm).

375


Page 25 of 33

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376

Cell viability study in spheroid cultures:

377

The BxPC-3 cell spheroids were prepared by seeding the 2 x 106 cells in each of the 96 well agarose

378

scaffolds. To make the scaffold, an aqueous agarose solution (2% W/V) was sterilized in an

379

autoclave for 45 minutes. The mold was purchased from Microtissues, and the scaffolds were

380

prepared using manufacturer’s protocol. The cells were seeded on 36 scaffolds and were incubated

381

for 7 days. Media on the scaffolds was changed every 2 days. The scaffolds were then divided

382

equally into two groups (18 scaffolds per group). One group of scaffold was allowed to grow under

383

normoxic conditions, and another group was incubated in a hypoxic chamber maintained at an

384

oxygen level of 1% (hypoxic conditions) for 24 hours. After establishing normoxic and hypoxic

385

cultures, the scaffolds were treated with gemcitabine, gemcitabine encapsulated hypoxia-sensitive

386

LNs, gemcitabine encapsulated iRGD functionalized hypoxia responsive LNs, and gemcitabine

387

encapsulating LNs devoid of iRGD and stimuli responsive lipids. Treatment with buffer and buffer

388

encapsulating LNs was used as a control. The scaffolds were divided equally into each of these

389

treatment groups (3 scaffolds in each treatment group). The cell spheroids were treated under

390

normoxic and hypoxic conditions for 72 hours. After the treatment, the cells in each spheroid

391

scaffold were dissociated by using a Tryple solution (1 mL for each scaffold) for 10 minutes. The

392

scaffolds were then washed with 3 mL of cell culture medium to harvest the cells. The harvested

393

cells from each scaffold were then dissociated and plated directly in 6 wells of 96 well plate. This

394

step converted the 3-dimensional cell culture to a monolayer culture. It was crucial to keep the

395

dilution of the cells exactly same for each treatment group to keep the relative ratios of cell viability

396

in each treatment group. The cell viability was then measured by the Alamar Blue assay.

397 398



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Page 26 of 33

399

Cellular uptake in spheroid cultures:

400

To facilitate the hypoxic microenvironment of tumors, we cultured the BxPC-3 cells as layers. To

401

grow the layered cells, a stack of wet strengthened Whatman filter paper (No. 114) was used.23

402

This setup allowed us to image the depth of penetration of the iRGD peptide-decorated LNs. We

403

cut these papers as 1” diameter circles using a commercially available hole punch. The cut circles

404

were then wrapped in aluminum foil and were autoclaved for 45 minutes. We designed a culture

405

apparatus (using the CAD software Creo Parametric) to hold the paper stack in place. The assembly

406

was then 3D printed using poly(lactic acid) as the “ink”. Our apparatus had four components –

407

base, paper stack insert, paper press insert attached to a media transfer tube, and a cover (Figure

408

8).

409


Page 27 of 33

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410 411

Figure 8. (A) The 3D printed parts for the cell culture apparatus to hold the stacks of Whatman

412

filter paper. (B) (a) The sterilized filter papers were inoculated with BxPC-3 cells embedded in

413

agarose and sodium alginate, (b) stacked with other filter papers, and (c) incubated together for

414

layered cell culture. The paper stack inert holds the cells inoculated filter papers. The paper insert

415

press allows holding the paper stack in place. The hollow tube inside the paper press insert allows

416

addition of culture media to the growing cells without disturbing the cell layers. These inserts were

417

contained in a chamber with the help of base and cover.

418

The trypsinized BxPC-3 cells were centrifuged, and 100,000 cells were suspended in 500 µL

419

culture medium. A solution of agarose and sodium alginate (1:2) was autoclaved, cooled to 40 °C,

420

and the cells suspension was added. After mixing, 20 µL of this suspension was applied at the



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Page 28 of 33

421

center of each paper circle. The cell-seeded papers were stacked together on the 3D printed paper

422

stack insert with the paper press insert on the top. The assembly was inserted in the base, and the

423

cover was placed. Two apparatus were then incubated under normoxic conditions, and two were

424

incubated in 1% oxygen for three days. The cell stacks in normoxic and hypoxic incubators

425

received treatments with 20 µL of carboxyfluorescein encapsulated LNs (delivered through the

426

hollow tube of the paper press insert) with and without the iRGD on the surface. The cell stacks

427

were treated for 2 hours in respective incubation conditions (normoxic/hypoxic), the paper racks

428

were removed, and were submerged in HBSS solution to wash excess of carboxyfluorescein. After

429

washing the stacks 3 times, they were again immersed in cell culture medium before imaging. To

430

image the cells with a fluorescence microscope, the papers from the stack were peeled one at a

431

time and fluorescence was measured in each paper layer. The fluorescence intensity and the depth

432

of penetration of polymersomes were measured using an Olympus laser scanning confocal

433

microscope. The images were analyzed with the ImageJ software.

434

AUTHOR INFORMATION

435

Corresponding Author

436

*Sanku Mallik, Department of Pharmaceutical Sciences, North Dakota State University, Fargo,

437

North Dakota 58102. Phone: 701-231-7888; Fax: 701-231-7831;

438

Email: [email protected]

439

Author Contributions

440

The manuscript was written through contributions of all authors. All authors have given approval

441

to the final version of the manuscript.


Page 29 of 33

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442

ACKNOWLEDGMENT

443

This research was supported by NSF grant DMR 1306154, and NIH grant 1 R01GM 114080 to

444

SM. PK was supported by a Doctoral Dissertation Award (IIA-1355466) from the North Dakota

445

EPSCoR (National Science Foundation).

446

SUPPORTING INFORMATION IS AVAILABLE

447

The Supporting Information is available free of charge at the ACS Publications website at DOI:

448

MALDI mass spectrum of hexynoic acid conjugated iRGD, CD Spectra of hexynoic acid

449

conjugated iRGD peptide before and after treatment with glutathione (GSH), CD spectrum

450

of iRGD functionalized DSPE lipid, release profile of the control DSPC liposomes (PDF).

451 452

References:

453

(1)

454 455

responsive nanocarriers for drug and gene delivery. J. Control. Release 126, 187-204. (2)

456 457

Ganta, S., Devalapally, H., Shahiwala, A., and Amiji, M. (2008) A review of stimuli-

Sharma, A., and Sharma, U. S. (1997) Liposomes in drug delivery: progress and limitations. Int. J. Pharm. 154, 123-140.

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O’brien, M., Wigler, N., Inbar, M., Rosso, R., Grischke, E., Santoro, A., Catane, R.,

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Kieback, D., Tomczak, P., and Ackland, S. (2004) Reduced cardiotoxicity and comparable

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efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX™/Doxil®)

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Oncol. 15, 440-449.

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Torchilin, V. (2011) Tumor delivery of macromolecular drugs based on the EPR effect. Adv. Drug Deliv. Rev. 63, 131-135.



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Danhier, F., Feron, O., and Préat, V. (2010) To exploit the tumor microenvironment:

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passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Control.

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Shabb, J. B., Padi, S. K., Singh, R. K., and Borowicz, P. P. (2014) MMP-9 Responsive

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PEG cleavable nanovesicles for efficient delivery of chemotherapeutics to pancreatic

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Höckel, M., and Vaupel, P. (2001) Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J. Natl. Cancer Inst. 93, 266-276.

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Vaupel, P., Kallinowski, F., and Okunieff, P. (1989) Blood flow, oxygen and nutrient

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supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 49,

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Brown, J. M., and Giaccia, A. J. (1998) The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res. 58, 1408-1416.

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Kizaka-Kondoh, S. (2015) Cell penetrating peptides improve tumor delivery of cargos

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Longati, P., Jia, X., Eimer, J., Wagman, A., Witt, M.-R., Rehnmark, S., Verbeke, C.,

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Toftgård, R., Löhr, M., and Heuchel, R. L. (2013) 3D pancreatic carcinoma spheroids

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induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing.

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Kiyose, K., Hanaoka, K., Oushiki, D., Nakamura, T., Kajimura, M., Suematsu, M.,

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Nishimatsu, H., Yamane, T., Terai, T., and Hirata, Y. (2010) Hypoxia-sensitive fluorescent

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Egawa, H., and Furusawa, K. (1999) Liposome adhesion on mica surface studied by atomic force microscopy. Langmuir 15, 1660-1666.



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Whitesides, G. M. (2009) Paper-supported 3D cell culture for tissue-based bioassays. Proc.

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TOC Graphic:

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